From larissa_yuri from yahoo.com.br Wed Apr 1 10:03:17 2009 From: larissa_yuri from yahoo.com.br (larissa yuri) Date: Wed Apr 1 12:22:22 2009 Subject: RNA extraction from human adipose tissue Message-ID: <161724.89044.qm@web54504.mail.re2.yahoo.com> Dear Shelley, ? I'm a scientist of State University of Campinas (UNICAMP), in Brazil, and our laboratory have researched human isolated adipose tissue and its glucose metabolism during stress. I very would like to use molecular biology, specificaly the Real-Time PCR technique, to quantify adipocytes enzimes (whole?tissue) and ?receptors (isolated adipocytes). But I didn't find a protocol of RNA extraction specific?to adipose tissue. Therefore, I ask you, please, the protocol of RNA Extraction in adipose tissue. I'll write you about my results. ? Thanks for your attention ? Larissa Yuri ? Department of Physiology and Biophysics, ?Institute of Biology (home page: www.ib.unicamp.br), State University of Campinas (UNICAMP), Campinas, Brazil Veja quais s?o os assuntos do momento no Yahoo! +Buscados http://br.maisbuscados.yahoo.com From lautys from gmail.com Wed Apr 1 19:58:38 2009 From: lautys from gmail.com (lautys) Date: Wed Apr 1 23:10:34 2009 Subject: RNA extraction from human adipose tissue In-Reply-To: <161724.89044.qm@web54504.mail.re2.yahoo.com> References: <161724.89044.qm@web54504.mail.re2.yahoo.com> Message-ID: i never deal with any adipose tissue; but as far as i concern, some stem cell research researchers are moving to adipose tissue, u might want to try to find the method in their area. Good luck. regards, Lau Tyan Shi On Wed, Apr 1, 2009 at 11:03 PM, larissa yuri wrote: > Dear Shelley, > > I'm a scientist of State University of Campinas (UNICAMP), in Brazil, and > our laboratory have researched human isolated adipose tissue and its glucose > metabolism during stress. I very would like to use molecular biology, > specificaly the Real-Time PCR technique, to quantify adipocytes enzimes > (whole tissue) and receptors (isolated adipocytes). But I didn't find a > protocol of RNA extraction specific to adipose tissue. Therefore, I ask you, > please, the protocol of RNA Extraction in adipose tissue. I'll write you > about my results. > > Thanks for your attention > > Larissa Yuri > > Department of Physiology and Biophysics, > Institute of Biology (home page: www.ib.unicamp.br), > State University of Campinas (UNICAMP), Campinas, Brazil > > > > Veja quais s?o os assuntos do momento no Yahoo! +Buscados > http://br.maisbuscados.yahoo.com > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From L.Kamalian from liverpool.ac.uk Thu Apr 2 04:23:57 2009 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Thu Apr 2 11:29:20 2009 Subject: Actin expression in protein extracted from tissue Message-ID: Hi, I have encountered a few problems after extracting protein from tissue sections. The tissue sections I used were primary tumour sections produced in mouse using sub-coetaneous injection of a lung malignant cell line. The sections were kept in RNA-later solution for 48 hours, then the solution was removed and the sections were transferred to - 80 freezer. To extract protein, the sections were cut into small pieces, then after adding lysing buffer (Cell-lytic M) they were sonicated 3 X 30 seconds. This followed by 30 mins centrifuge, full speed and then the supernatant was removed and the protein concentration was measured by Bradford Assay. All samples showed quite good protein content (3-8 mg/ml). So far so good. Then the problem started: to confirm the protein concentration and also the quality of the samples, 30 ug of each were subjected to polyacrylamide gel electrophoresis, followed by western blot, using mouse anti b-actin mono-clonal antibody as the primary and rabbit anti mouse as the secondary, with the usual dilutions and incubation period. The result is very strange. First of all there are lots of non-specific bands which have never occurred to any of other actin blots before. Secondly, the assumed actin band shows extreme difference in protein loaded. Looking at the gel after transfer, there is not that much difference between the samples. Moreover, the other nonspecific bands are equal!! I am lost. Please help me. L. From eenigoy from gmail.com Thu Apr 2 05:30:26 2009 From: eenigoy from gmail.com (yoginee budhkar) Date: Thu Apr 2 11:29:45 2009 Subject: dA extension of PCR product for TA cloning Message-ID: <77ddf6c70904020330o54af6a0dte15f6ee3ae2274a4@mail.gmail.com> Dear All, Is it okay to simply replenish the contents like taq buffer and taq polymerase in already prepared and stored PCR product (which is not purified) to carry out dA extension so that the product becomes useful for TA cloning? This is what I plan to do: 43 ul of PCR product (not purified, hence already contains salts n other buffer components) 5 ul of 10X Taq buffer (fresh) 1 ul Taq pol (1U) 1 ul of dATP (0.5mM final conc) 50 ul (total volume) incubate at 72degree C for 30 min Will the already present buffer components and salts in the PCR reaction mix hamper taq pol activity? -- Yg From novalidaddress from nurfuerspam.de Thu Apr 2 12:25:14 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Apr 2 13:28:52 2009 Subject: Actin expression in protein extracted from tissue References: Message-ID: <31f47cf8-2240-403d-bbfc-8f1f4e73965a@z15g2000yqm.googlegroups.com> Dear Laleh, the solubilization of (polymerized!) actin dependes largely on the detergents present in your lysis buffer. As long there is no SDS present, I'd expect actin in the pellet. I am deducting this from the fact that one may use buffers with eg Triton X100 or Tween 20 to nicely dissolve adherent cells but keep nuclei and the cytoskeleton attached to the well. You might easily compare your lysis buffer with a lysate obtained directly by sonication of your sample in 1x Laemmli buffer. Have fun Wolfgang From pjie2 from cam.ac.uk Thu Apr 2 11:42:38 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Thu Apr 2 13:28:58 2009 Subject: dA extension of PCR product for TA cloning In-Reply-To: References: Message-ID: Why would you want to add more of the buffer salts? It's not like magnesium, sodium and chloride ions can "go off" over time. In theory you could just add fresh nucleotides (in case the original PCR reaction depleted them too severely) and some Taq. However, ask yourself this question: why are you doing an A-tailing reaction? Presumably the answer is because the polymerase you used to do the PCR either does not add A-tails, or (likely for many polymerase mixes) it has 3' to 5' exonuclease activity and in fact removes them. In the latter case, then there's no point adding A-tails without purifying it, as any remaining enzyme from the initial PCR will just chew the tails off again. Purify it before you A-tail it - it only adds a few minutes and takes all the unknowns out of the equation. Peter On Thu, 2 Apr 2009, yoginee budhkar wrote: > Dear All, > > Is it okay to simply replenish the contents like taq buffer and taq > polymerase in already prepared and stored PCR product (which is not > purified) to carry out dA extension so that the product becomes useful for > TA cloning? > > This is what I plan to do: > > 43 ul of PCR product (not purified, hence already contains salts n other > buffer components) > 5 ul of 10X Taq buffer (fresh) > 1 ul Taq pol (1U) > 1 ul of dATP (0.5mM final conc) > 50 ul (total volume) > > incubate at 72degree C for 30 min > > Will the already present buffer components and salts in the PCR reaction mix > hamper taq pol activity? > > -- Yg > From davidminde from gmail.com Thu Apr 2 13:34:06 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Thu Apr 2 15:25:23 2009 Subject: dA extension of PCR product for TA cloning In-Reply-To: <77ddf6c70904020330o54af6a0dte15f6ee3ae2274a4@mail.gmail.com> References: <77ddf6c70904020330o54af6a0dte15f6ee3ae2274a4@mail.gmail.com> Message-ID: <123243900904021134g300da85an5beb03eea400fde7@mail.gmail.com> No, (unless you are using a low-quality polymerase with bad proofreading;) best, David 2009/4/2 yoginee budhkar > Dear All, > > Is it okay to simply replenish the contents like taq buffer and taq > polymerase in already prepared and stored PCR product (which is not > purified) to carry out dA extension so that the product becomes useful for > TA cloning? > > This is what I plan to do: > > 43 ul of PCR product (not purified, hence already contains salts n other > buffer components) > 5 ul of 10X Taq buffer (fresh) > 1 ul Taq pol (1U) > 1 ul of dATP (0.5mM final conc) > 50 ul (total volume) > > incubate at 72degree C for 30 min > > Will the already present buffer components and salts in the PCR reaction > mix > hamper taq pol activity? > > -- Yg > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) From novalidaddress from nurfuerspam.de Thu Apr 2 15:49:58 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Apr 2 19:00:16 2009 Subject: dA extension of PCR product for TA cloning References: Message-ID: <4d4561d1-c7fb-4681-8cf9-451e395063c9@z1g2000yqn.googlegroups.com> On Apr 2, 12:30?pm, yoginee budhkar wrote: > Dear All, > > Is it okay to simply replenish the contents like taq buffer and taq > polymerase in already prepared and stored PCR product (which is not > purified) to carry out dA extension so that the product becomes useful for > TA cloning? > > This is what I plan to do: Hi, you could leave out the second aliquot of Taq buffer. If paranoid, add a bit of DTT. Wo > > 43 ul of PCR product (not purified, hence already contains salts n other > buffer components) > 5 ul of 10X Taq buffer (fresh) > 1 ul Taq pol (1U) > 1 ul of dATP (0.5mM final conc) > 50 ul (total volume) > > incubate at 72degree C for 30 min > > Will the already present buffer components and salts in the PCR reaction mix > hamper taq pol activity? > > -- Yg From pjie2 from cam.ac.uk Thu Apr 2 17:31:42 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Thu Apr 2 19:00:24 2009 Subject: Actin expression in protein extracted from tissue In-Reply-To: References: Message-ID: <73ksmuFvicquU1@mid.individual.net> DK wrote: > In article , "Kamalian, Laleh" wrote: >> Hi, I have encountered a few problems after extracting protein from tissue = >> sections. The tissue sections I used were primary tumour sections produced= >> in mouse using sub-coetaneous injection of a lung malignant cell line. The= >> sections were kept in RNA-later solution for 48 hours, > > Since you are using a solution for which composition is unknown, > it is impossible to say what might have happened to the cells/proteins > in your samples during the storage... RNALater is largely an extremely concentrated ammonium sulphate solution. It preserves RNA by precipitating out the RNAses in the tissue. Without knowing the composition of your lysis buffer, it's hard to say whether you would have re-solubilised the actin in the sample. Peter From L.Kamalian from liverpool.ac.uk Fri Apr 3 05:46:12 2009 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Fri Apr 3 11:26:30 2009 Subject: Actin expression in protein extracted from tissue Message-ID: Hi. Thanks to every body who responded. The reason I kept the tissue in RNAlater was that they were supposed to be used for RNA extranction. However my supervisor asked me to extract protein out of them. The information leaflet came with the solution claims that the solution doesn't touch the protein and they are safe. I also double check this with their costomer services and they said the same thing. The CellLytic-M is the lysis buffer that we have been using to extract protein from our cell-lines and it has been serving us well. The centrifuge speed was 13000 rpm. Above all this method of extraction was previously done by one of my colleagues and worked very well, it gave a single band of b-actin as the normalizer. I followed her instructions exactly. I tried mouse monoclonal anti GAPDH yesterday and it also gave me nonspecific bands, although the actual band ( according to the size) was much better than the actin's. So is there any further thoughts? Thanks L. From pjie2 from cam.ac.uk Fri Apr 3 12:23:15 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Fri Apr 3 15:47:11 2009 Subject: Actin expression in protein extracted from tissue In-Reply-To: References: Message-ID: On Fri, 3 Apr 2009, Kamalian, Laleh wrote: > > Above all this method of extraction was > previously done by one of my colleagues and worked very well, it gave a > single band of b-actin as the normalizer. I followed her instructions > exactly. I tried mouse monoclonal anti GAPDH yesterday and it also gave > me nonspecific bands, although the actual band ( according to the size) > was much better than the actin's. So is there any further thoughts? Grab some tissue you have plenty of, soak it in RNALater and practice the extraction protocol until it works in your hands before moving on to your precious tumour samples. Do be aware that RNALater-treated samples are only suitable for *denaturing* gels and applications. The information leaflet is quite clear on this point. Peter From bb-bb from gazeta.pl Fri Apr 3 14:47:06 2009 From: bb-bb from gazeta.pl (Basia Dymek) Date: Fri Apr 3 15:47:17 2009 Subject: Expression of protein - CMV promoter Message-ID: Hi! I have prepared a plasmid using pFLAG-CMV-2 vector (Sigma) into which I have cloned my gene of interest. Finally, I have some level of expression after transfection HepG2 cells, however it is unsatisfactory for me. CMV promoter should be the strong one. Have you got any clues what could be the reason of low level of expression of my protein? I would be thankful for any advice. Basia From shifalich from rediffmail.com Fri Apr 3 23:24:01 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat Apr 4 11:34:50 2009 Subject: Expression of protein Message-ID: <20090404042401.9837.qmail@f4mail-234-240.rediffmail.com> ? Hi all! I also have expression problem. I tried expressing my protein, cDNA cloned in pET19b in Rosetta gami cells ( beacause it has 5 disulphide bonds and cDNA is from snake)and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation) Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from expression hosts. DNA is there but no protein. Any comments??????? On Sat, 04 Apr 2009 DK wrote : >In article , Basia Dymek wrote: > >Hi! > >I have prepared a plasmid using pFLAG-CMV-2 vector (Sigma) into which I have > >cloned my gene of interest. Finally, I have some level of expression after > >transfection HepG2 cells, however it is unsatisfactory for me. > >Unfortunately, when it comes to overexpression in mammalian cells, >the typical protein levels are unsatisfactory to most... > > >CMV promoter > >should be the strong one. Have you got any clues what could be the reason of > >low level of expression of my protein? I would be thankful for any advice. > >The vector is for transient expression. Thus, if your transfection is >inefficient, expression level will be low no matter what. Make sure >you transfect at least 50% of cells. Lots of things involved, starting >with s high quality plasmid prep and ending with the best transfection >method. Beyond that: > >Besides the strenth of promoter, many factors affect expression levels: >rate of transcription is influenced by the gene's sequence, mRNA stability >varies, rate of translation may be influenced by mRNA sequence/structure, >and protein stability varies quite a bit. In addition, overexpression of some >proteins is toxic to cells and inhibits transcription/translation machinery. >Most of these you can't change rationally, so just accept a fact of life that >some proteins express at higher levels than others. > >DK >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From Sharon.Waldrop from utsouthwestern.edu Sun Apr 5 20:24:33 2009 From: Sharon.Waldrop from utsouthwestern.edu (Sharon Waldrop) Date: Sun Apr 5 21:32:04 2009 Subject: Coulter Multisizer II Cell Counter Message-ID: <49D913810200003E0004664E@swnw126.swmed.org> Hello Everyone, When we moved labs, some how the User Manual (Coulter Multisizer II Cell Counter) did not make it. I have contacted the old lab - they cannot find it. I have contacted Coulter - they no longer have this manual and have not for some time, it seems. Does any one out there have the user manual to this piece of equipment? Any help would be appreciated. Thanks, Shar From bmacgreg from unc.edu Tue Apr 7 10:30:09 2009 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Tue Apr 7 10:45:07 2009 Subject: Coulter Multisizer II In-Reply-To: <200904061705.n36H5Rh01966@net.bio.net> References: <200904061705.n36H5Rh01966@net.bio.net> Message-ID: <9D9BD91B-9DF2-40A7-AF7C-42A00FB64244@unc.edu> Hi Sharon, Google knows all, this was the second hit after your query: www.ibb.gatech.edu/facilities/confocal/pdf/CoulterMultisizerII.pdf Hope that's what you were looking for. Enjoy! Barbara From timegg113 from gmail.com Tue Apr 7 15:05:39 2009 From: timegg113 from gmail.com (Tim Eggleston) Date: Tue Apr 7 16:15:46 2009 Subject: mice microfil injection Message-ID: Hello, We are going to do a microfil injection in the near future and are in desparate need for information. The microfil needs to be injected in the abdominal aorta of a mouse. We have never done this, and any information would be helpful. I have found some information on PubMed but it is mostly from the 70's and 80's and not very thorough. Thanks! Tim Eggleston Tim Eggleston Research Assistant II Iowa Comprehensive Lung Imaging Center Dept. of Radiology University of Iowa 319-384-7926 From qiz from upmc.edu Wed Apr 8 09:09:17 2009 From: qiz from upmc.edu (Qi, Zengbiao) Date: Wed Apr 8 11:38:25 2009 Subject: transgene expression problem Message-ID: <7F246E57CDE48245AE8CA3DA231CD8BA230528DF@msxmbxnsprd04.acct.upmchs.net> I have developed a transgenic mouse strain expressing HES1 in CD4 positive T cells by utilizing Tet repressor system. The mouse is double positive for M2 (under CD4 promoter) and HES1(under Tet repressor control) and feeding the double positive mice with food containing doxycycline (1g/kg) induces HES1 expression in CD4+ T cells in both thymus and spleens, at least, theoretically. However, HES1 is only induced in thymi, not in spleens of my transgenic mice. Although HES1 can be induced in isolated splenic CD4+ T cells in vitro culture with doxycycline, even without any stimulation in 24 hrs. I’d appreciate your insight of the problem. Zengbiao From yvonne.couch from pharm.ox.ac.uk Fri Apr 10 12:13:41 2009 From: yvonne.couch from pharm.ox.ac.uk (Yvonne Couch) Date: Fri Apr 10 13:23:42 2009 Subject: Question... Message-ID: <001301c9b9ff$b2e2d920$18a88b60$@couch@pharm.ox.ac.uk> I have an odd question for everyone which I hope might bring up a nice variety of responses; you have the 20 amino acid sequence of a peptide you believe to be a new GPCR, what techniques would you use to find out its function? From Victor.Emelyanov from newcastle.ac.uk Sun Apr 12 12:44:12 2009 From: Victor.Emelyanov from newcastle.ac.uk (Victor Emelyanov) Date: Sun Apr 12 14:30:08 2009 Subject: Cytochrome C western blot Message-ID: Hi Using Mab against cyt c (Santa-Cruz, 7H8 clone) in WBA of mitochondrial fractions, I always oserve only band of ca. 70 kDa and never innate protein of 15 kDa. Long ago, I saw the same using Pharmingen Mab. At that time I was said that cyt c forms covalently linked complex with some specific protein and that even relevant paper exists. Did you fix a problem? Indeed covalent complex? Or multimer? Now and then I didn't use chaotropic agent in Laemmli sample buffer (8M urea or Guanidine chloride). Maybe this is important? Don't hesitate to contact me. Best, Dr Victor V. Emelyanov, PhD Senior Research Associate Marie Curie Fellow Institute for Cellular & Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, United Kingdom From mgbiotec from gmail.com Sun Apr 12 22:06:15 2009 From: mgbiotec from gmail.com (megha goyal) Date: Sun Apr 12 23:54:33 2009 Subject: Chemically defined media required Message-ID: we are currently producing our protein using e.coli w3110 containing puc 18 plasmid having the gene of interest. our protein is temperature inducible i.e we grow our culture at 30? C and induction is at 42 ? C. our protein is expressed as Inclusion bodies. we are using complex medium having yeast extract and tryptone with addtion of trace metals and vitamins during fermentation. glucose is added in initital stage as well as fed intermittently as per the requirement mostly durign the induction. the culture grows to an O.D. of 10 [6hrs] and then we induce it [2hrs] this is as per our technology providers advice. we would like to switch over to chemically defined media from complex media so as to increase yield. can anyone suggest how can we go about it as the media constituent we found on the net gives trace metals content to be added but not of vitamins. kindly help. biotef From davidminde from gmail.com Mon Apr 13 02:48:04 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Mon Apr 13 10:54:54 2009 Subject: Chemically defined media required In-Reply-To: References: Message-ID: <123243900904130048y1bd3d735w600ce36df3473ebb@mail.gmail.com> M9 (probably THE standard chemically defined medium in maniatis) does not give higher growth than about OD 5 in simple erlenmeyer. If really maximum volumetric yield is an aim, use a fermenter and (fed-batch and) rich media or (if not available) just terrific broth in batch. best, David (PS never heard of induction @ 42, funny system ... we rather like it cool for making [very functional:] protein) 2009/4/13, megha goyal : > we are currently producing our protein using e.coli w3110 containing puc 18 > plasmid having the gene of interest. our protein is temperature inducible > i.e we grow our culture at 30? C and induction is at 42 ? C. our protein is > expressed as Inclusion bodies. > > we are using complex medium having yeast extract and tryptone with addtion > of trace metals and vitamins during fermentation. glucose is added in > initital stage as well as fed intermittently as per the requirement mostly > durign the induction. the culture grows to an O.D. of 10 [6hrs] and then we > induce it [2hrs] this is as per our technology providers advice. > > we would like to switch over to chemically defined media from complex media > so as to increase yield. can anyone suggest how can we go about it as the > media constituent we found on the net gives trace metals content to be added > but not of vitamins. kindly help. > > biotef > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) From aawara from pontiff-playground.org Mon Apr 13 09:13:28 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Apr 13 10:55:16 2009 Subject: Chemically defined media required References: Message-ID: In , DK wrote: >>we would like to switch over to chemically defined media from complex media >>so as to increase yield. can anyone suggest how can we go about it as the >>media constituent we found on the net gives trace metals content to be adde= >>d >>but not of vitamins. kindly help. First off, it is highly unlikely that a chemically defined medium, such as M9 minimal media, suggested by DK will increase yield, which appears to be your primary concern - as opposed to increasing yield - unless by the latter you mean soluble protein. For solubility, I agree with DK's suggestion that you should switch to a chemical inducer, rather than temperature induction - what are you using - a ts mutant of lambda cI? That system, now several years old, typically placed two copies of lambda OR1 immediately after the tac promoter or the lacUV5 promoter. At 30oC, cI was bound to the operators blocking the elongation of transcripts that initiated at tac or lacUV5; at 42oC cI was inactivated, permitting transcript elongation. While the lacUV5 and tac promoters are strong promoters for E. coli RNAP, they are weak compared to the activity of T7 RNAP on the T7 promoter. So re-cloning your protein into one of Bill Studier's pET vectors, and inducing them in a strain that inducibly expresses T7 RNAP, such as BL21(DE3) - or one of its many derivatives, will significantly increase your yield. In addition, as Dima points out, you can induce at much lower temperatures, which in turn will likely increase solubility. > Google "M9 minimal medium". If the protein is insoluble, heat induction > is likely not a way to go. You'd be better off switching to IPTG induction > and expression at 16C. Vitamins are not required for a typical expression > strain. Yes - this is correct. One other suggestion. What about just denaturing and renaturing the protein that is in inclusion bodies? AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From davidminde from gmail.com Tue Apr 14 04:20:19 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Tue Apr 14 09:56:04 2009 Subject: Chemically defined media required In-Reply-To: References: Message-ID: <123243900904140220g1394a833x5258ad59aedc3b32@mail.gmail.com> > Yet some refold very easily. It's always, always worth a shot. > Easy to do, nothing to lose. > I slightly disagree: For proteins where NO rock solid functional test is > available for proper validation of refolding success, the risk of ending up > with a heterogeneous population of partly refolded and partly misfolded > protein is significantly increased by refolding (vs. e.g. soluble expression > under cold-shock conditions) making it completely useless for most (ensemble > based) biophysical and structure biology assays. cheers, > > David > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Science is what happens while we are making other plans (~John Lennon) From Paul.Phelan from tufts.edu Wed Apr 15 12:47:56 2009 From: Paul.Phelan from tufts.edu (Paul J. Phelan) Date: Wed Apr 15 13:40:16 2009 Subject: Separating GST from GST-fusion protein Message-ID: <20090415134756.yfgavl8c0s0owk8s@webmail.tufts.edu> I think I asked this question before on this forum, but I thought I'd try it again just in case anyone has any new "tricks" or ideas, because I'm having the same old problem again. I am trying to purify a GST-fusion protein, and it's contaminated with GST. The GST-fusion has a mol. wt. of 40,000 and GST is 26,000, but on a Sephacryl S100 gel filtration column, both proteins are co-eluting in a single, beautiful peak. Anyone would think that such a textbook peak would only contain pure protein, but no - half of it is GST! I even re-ran the gel filtration column in a buffer containing 0.4 M NaCl to try to separate the two proteins, but I got the same single peak, so the GST must be still there. Now I am not talking about digesting a GST-fusion and then removing GST; I have no problem doing that, but I am trying to purify some intact GST-fusion, without taking GST with it: does anyone have a magic trick to get rid of GST? Thank you Paul Phelan From hengen from earthlink.net Wed Apr 15 14:43:06 2009 From: hengen from earthlink.net (hengen@earthlink.net) Date: Wed Apr 15 15:47:55 2009 Subject: Lab Tech Articles at BiteSizeBio Message-ID: <4b649e78-e998-456f-8c41-740518c81677@w35g2000prg.googlegroups.com> For all you bionetters interested in old school lab techniques, I've posted my list of all-time favorites from the days when I was active on the methods and reagents newsgroup and writing tech articles about the list topics. I'm now writing more of the same kind of articles for BiteSizeBio at http://bitesizebio.com/ The newest article can be found at http://bitesizebio.com/2009/04/13/low-tech-lab-gadgets-and-solutions-my-all-time-favs/ If you like the article and want to see more like this, or have an idea about what you would like me to write about, please send email to hengen_at_earthlink.net I'm also interested to hear back from my old friends on the methods and reagents newgroup. Feel free to drop me an email. Paul N. Hengen, Ph.D. http://www.linkedin.com/in/hengen From davidminde from gmail.com Thu Apr 16 03:47:32 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Thu Apr 16 10:41:59 2009 Subject: Chemically defined media required In-Reply-To: References: Message-ID: <123243900904160147y780176a1l3edbfe3a4858ca41@mail.gmail.com> ... if it forms x-tals, u are lucky ... which will not happpen (or result in crystals with high "invisible" fraction) for many most interesting uncharacterized parts of (un)foldome of human proteome :) Morover, strictly speaking, a crystal does NOT mean necessarily having the protein in a biologically active state ... ... Even though some nobel laureates still think so ;) best, David Science is what happens while we are making other plans (~John Lennon) From Jean-Luc.Gallois from avignon.inra.fr Thu Apr 16 03:12:33 2009 From: Jean-Luc.Gallois from avignon.inra.fr (Gallois) Date: Thu Apr 16 10:43:07 2009 Subject: methods@net.bio.net Message-ID: <49E6E871.1080601@avignon.inra.fr> Dear colleagues, as I want to do gateway cloning in a kan-resistant binary vector, I am looking for an entry vector with a non-kanamycin resistance. Ideally, this entry clone would look like a basic pENTR (as pENTR4 sold by invitrogen) with a good MCS between attL1 and attL2 but with a gentamicin resistance gene. Although invitrogen has designed both KanR and GentaR type of plasmids for pDONR (pDONR201 and pDONR207) I could not find a similar set of plasmids for pENTR. If anyone knows about such a plasmid or had designed one and would be likely to share it, I would be really grateful, Regards Jean-Luc Jean-Luc GALLOIS INRA-UR 1052 G?n?tique et Am?lioration des Fruits et L?gumes Dom. St Maurice, BP94 84143 Montfavet cedex, France From novalidaddress from nurfuerspam.de Thu Apr 16 01:59:44 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Apr 16 10:43:16 2009 Subject: Separating GST from GST-fusion protein References: Message-ID: <8e586110-9eaf-4b8f-bf8a-d8cd553cdcea@e18g2000yqo.googlegroups.com> Hi Paul, you might want to consider isoelectric focusing. For a nice inexpensive colection of protocols, see Shen 2008. Methods in Molecular Biology, vol. 424, Sample Preparation and Fractionation, Volume 1, 187-203. Best regards, Wo From novalidaddress from nurfuerspam.de Thu Apr 16 02:08:21 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Apr 16 10:43:25 2009 Subject: Lab Tech Articles at BiteSizeBio References: <4b649e78-e998-456f-8c41-740518c81677@w35g2000prg.googlegroups.com> Message-ID: Dear Paul, nice to see you back here! I am very happy to admit that you are "responsible" a great deal for my knowledge in molecular biology and biochemistry (I started as chemist a long time ago and dared to deal with cells, enzymes and metabolism for my PhD thesis), as to the methods NG, I was guided by your articles in TiBS. There barely was internet, Netscape just had become available and the Bionet bcame an ivaluable source of knowledge and learning. And it still is. I hope I am able to give back now a bit of what I got from the Bionet now and then. Many thanks! Wolfgang From Jean-Luc.Gallois from avignon.inra.fr Thu Apr 16 03:14:19 2009 From: Jean-Luc.Gallois from avignon.inra.fr (Gallois) Date: Thu Apr 16 10:43:35 2009 Subject: Non kanamycin-resistant pENTR vector Message-ID: <49E6E8DB.7040101@avignon.inra.fr> Dear colleagues, as I want to do gateway cloning in a kan-resistant binary vector, I am looking for an entry vector with a non-kanamycin resistance. Ideally, this entry clone would look like a basic pENTR (as pENTR4 sold by invitrogen) with a good MCS between attL1 and attL2 but with a gentamicin resistance gene. Although invitrogen has designed both KanR and GentaR type of plasmids for pDONR (pDONR201 and pDONR207) I could not find a similar set of plasmids for pENTR. If anyone knows about such a plasmid or had designed one and would be likely to share it, I would be really grateful, Regards Jean-Luc Jean-Luc GALLOIS INRA-UR 1052 G?n?tique et Am?lioration des Fruits et L?gumes Dom. St Maurice, BP94 84143 Montfavet cedex, France From pow.joshi from gmail.com Thu Apr 16 12:37:22 2009 From: pow.joshi from gmail.com (Pow Joshi) Date: Thu Apr 16 13:45:14 2009 Subject: Separating GST from GST-fusion protein In-Reply-To: <20090415134756.yfgavl8c0s0owk8s@webmail.tufts.edu> References: <20090415134756.yfgavl8c0s0owk8s@webmail.tufts.edu> Message-ID: <710764ea0904161037s17e202fao2eb8e7bd17884c83@mail.gmail.com> 2009/4/15 Paul J. Phelan : > I think I asked this question before on this forum, but I thought I'd try it > again just in case anyone has any new "tricks" or ideas, because I'm having > the same old problem again. I am trying to purify a GST-fusion protein, and > it's contaminated with GST. The GST-fusion has a mol. wt. of 40,000 and GST > is 26,000, but on a Sephacryl S100 gel filtration column, both proteins are > co-eluting in a single, beautiful peak. Anyone would think that such a > textbook peak would only contain pure protein, but no - half of it is GST! > I even re-ran the gel filtration column in a buffer containing 0.4 M NaCl > to try to separate the two proteins, but I got the same single peak, so the > GST must be still there. > Now I am not talking about digesting a GST-fusion and then removing GST; I > have no problem doing that, but I am trying to purify some intact > GST-fusion, without taking GST with it: does anyone have a magic trick to > get rid of GST? ah, only if it is absolutely imperative that you have a pure protein, and have some time at hand: perhaps you could generate polyclonals against the whole mixture of gst+ gst-protein; then drain out the gst antibodies against a gst column.... once that is done, and you are left with the antibodies against your protein, which you can then use to affinity purify your fusion .... Gee, but I guess it's far too involved anyways .... perhaps Dima's and Wo's ideas are better to try first.... best, Pow > > Thank you > > Paul Phelan > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From gerchman from research.haifa.ac.il Thu Apr 16 15:30:42 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Thu Apr 16 16:14:43 2009 Subject: Were to get a Yeda press? Message-ID: <1239913842.49e7957264aa1@webmail.haifa.ac.il> Greetings netters I am trying to find were to buy a Yeda press (hopefully cheaper than a french press). Does anyone know were one can find them? Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From Li from humacyte.com Thu Apr 16 14:28:16 2009 From: Li from humacyte.com (Yuling Li) Date: Thu Apr 16 16:14:52 2009 Subject: CMC for SDS? Message-ID: <4502DBE4D415DE418C6FD652DB4C501B05890B@humacyte-sbs.Humacyte.internal> Dear Shaun: Do you happen to know what's the CMC of SDS giving the concentration of NaCL of 1 M? Do you know someone who can measure CMC of SDS? I am looking forward to your answers. Sincerely, Yuling Yuling Li, MD, Ph.D. Humacyte, Inc. PO Box 12695 7020 Kit Creek Road, Suite 230 Research Triangle Park, NC 27709 Phone: 919.313.9633 ext.234 Cell: 919-225-1831 Fax: 919.313.9634 li@humacyte.com From zhiqi2 from illinois.edu Thu Apr 16 15:20:26 2009 From: zhiqi2 from illinois.edu (Zhi Qi) Date: Thu Apr 16 16:14:58 2009 Subject: A question related to the DNA hairpin reannealing when helicase binds on this hairpin Message-ID: <49E7930A.9080608@illinois.edu> Dear all, I have a question related to the DNA hairpin reannealing when helicase binds on this hairpin: I have a DNA hairpin construct (89-bp on the stem), and then I load helicase to monitor hairpin unwound. The helicase I studied can bind around 20-nt ssDNA. So my question is that if only one helicase unwound the hairpin completely, does the DNA reanneal behind the helicase as the helicase only use 20-nt position and the hairpin is 89-bp? I read some paper, and it seems like if the helicase binds on the hairpin, even only one helicase, then no matter how long the hairpin is, the hairpin can not reanneal until the helicase unbind the hairpin. Is that true? If anyone knows some conclusion or reference, please let me know. Thank you so much! Best Regards, Zhi Qi Biophysics, UIUC From davidminde from gmail.com Thu Apr 16 18:51:00 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Thu Apr 16 19:28:47 2009 Subject: CMC for SDS? In-Reply-To: <4502DBE4D415DE418C6FD652DB4C501B05890B@humacyte-sbs.Humacyte.internal> References: <4502DBE4D415DE418C6FD652DB4C501B05890B@humacyte-sbs.Humacyte.internal> Message-ID: <123243900904161651w59625902gac214c6f16cff4c2@mail.gmail.com> ... you could try googling for SDS Critical micellar concentration ;) There are no big secrets about that ... D 2009/4/16, Yuling Li : > Dear Shaun: > > > > Do you happen to know what's the CMC of SDS giving the concentration of > NaCL of 1 M? > > > > Do you know someone who can measure CMC of SDS? > > > > I am looking forward to your answers. > > > > Sincerely, > > > > Yuling > > > > > > > > Yuling Li, MD, Ph.D. > > Humacyte, Inc. > > PO Box 12695 > > 7020 Kit Creek Road, Suite 230 > > Research Triangle Park, NC 27709 > > Phone: 919.313.9633 ext.234 > > Cell: 919-225-1831 > > Fax: 919.313.9634 > > li@humacyte.com > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) From MConnelly from lab901.com Fri Apr 17 04:43:41 2009 From: MConnelly from lab901.com (Matthew Connelly) Date: Fri Apr 17 11:10:29 2009 Subject: CMC for SDS? In-Reply-To: <123243900904161651w59625902gac214c6f16cff4c2@mail.gmail.com> References: <4502DBE4D415DE418C6FD652DB4C501B05890B@humacyte-sbs.Humacyte.internal> <123243900904161651w59625902gac214c6f16cff4c2@mail.gmail.com> Message-ID: <5A6456E670F5564EB7095EC34563B39DC2B43A@lab901-svr01.Lab901.Com> CMC is dependent on ionic strength and can be difficult to predict accurately. Depending on how important it is that you know accurately or not, Bohuslav Gas' research group has done work on determining and predicting CMC of SDS under various conditions, including a very reliable method using system eigenmobilities by electrophoresis, which is better than the standard method of testing by conductivity. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of David-Paul Minde Sent: 17 April 2009 00:51 To: Yuling Li Cc: methods@magpie.bio.indiana.edu Subject: Re: CMC for SDS? ... you could try googling for SDS Critical micellar concentration ;) There are no big secrets about that ... D 2009/4/16, Yuling Li : > Dear Shaun: > > > > Do you happen to know what's the CMC of SDS giving the concentration of > NaCL of 1 M? > > > > Do you know someone who can measure CMC of SDS? > > > > I am looking forward to your answers. > > > > Sincerely, > > > > Yuling > > > > > > > > Yuling Li, MD, Ph.D. > > Humacyte, Inc. > > PO Box 12695 > > 7020 Kit Creek Road, Suite 230 > > Research Triangle Park, NC 27709 > > Phone: 919.313.9633 ext.234 > > Cell: 919-225-1831 > > Fax: 919.313.9634 > > li@humacyte.com > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht Science is what happens while we are making other plans (~John Lennon) _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From engelbert_buxbaum from hotmail.com Fri Apr 17 07:47:06 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Apr 17 11:10:40 2009 Subject: CMC for SDS? References: Message-ID: Am 16.04.2009, 15:28 Uhr, schrieb Yuling Li : > Do you happen to know what's the CMC of SDS giving the concentration of > NaCL of 1 M? You'll have to measure that, as the cmc is ionic strength dependent. The easiest way to do that is described in: @ARTICLE{Cha-84, AUTHOR= {A. Chattopadhyay and E. London}, TITLE= {Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge}, JOURNAL={Anal. Biochem.}, YEAR= {1984}, VOLUME= {139}, PAGES= {408-412} } From pjie2 from cam.ac.uk Fri Apr 17 12:14:58 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Fri Apr 17 16:38:44 2009 Subject: Separating GST from GST-fusion protein In-Reply-To: References: <20090415134756.yfgavl8c0s0owk8s@webmail.tufts.edu> Message-ID: On Thu, 16 Apr 2009, Pow Joshi wrote: > > ah, only if it is absolutely imperative that you have a pure protein, > and have some time at hand: perhaps you could generate polyclonals > against the whole mixture of gst+ gst-protein; then drain out the gst > antibodies against a gst column.... once that is done, and you are > left with the antibodies against your protein, which you can then use > to affinity purify your fusion .... Gee, but I guess it's far too > involved anyways .... perhaps Dima's and Wo's ideas are better to try > first.... It looks as though the converse might make more sense. If there's an antibody available that recognises free GST but not a GST fusion, you could use that to remove the free GST from your mix. Unfortunately I don't know if any such antibody exists! Would be nice if there is one, since it's a general solution to the problem and should work no matter what fusion partner you're working with. Peter From Lionel.Brooks from dartmouth.edu Sat Apr 18 14:57:10 2009 From: Lionel.Brooks from dartmouth.edu (Lionel Brooks 3rd) Date: Sat Apr 18 18:47:20 2009 Subject: Sodium Orthovanadate Preparation Message-ID: <49EA3096.3080107@dartmouth.edu> Hello All, I'm trying to prepare a 100 mM stock solution of sodium orthovanadate. I want to use vanadate in my lysis buffer. I think this is a pretty standard practice. I tried following instructions found on the Abcam website: Sodium orthovanadate preparation: This needs to be done under the fume hood ? Prepare a 100 mM solution in double distilled water ? Set pH to 9.0 with HCl ? Boil until colorless ? Cool to room temperature ? Set pH to 9.0 again ? Boil again until colorless ? Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling ? Bring up to the initial volume with water ? Store in aliquots at -20 ?C Much to my chagrin the solution is not colorless after more than 30 min of boiling. Any ideas on what I am doing wrong? Appreciatively, Lee From ykfa from deakin.edu.au Mon Apr 20 01:03:52 2009 From: ykfa from deakin.edu.au (ykfa@deakin.edu.au) Date: Mon Apr 20 09:29:41 2009 Subject: RNA extraction Message-ID: <20090420160352.dgw3a6vvfo4cok0o@www.deakin.edu.au> Hello, Just wondering if there is any way to extract RNA, prefer just the rRNA, of e. coli without losing the proteins? Yvonne Yvonne Fazendin, PhD Candidate Deakin University, Pigdons Rd Geelong Victoria 3217 Australia. Phone: 03 5227 2758 International: +61 3 5227 2758 Email: ykfa@deakin.edu.au Website: http://www.deakin.edu.au Deakin University CRICOS Provider Code 00113B (Vic) Important Notice: The contents of this email are intended solely for the named addressee and are confidential; any unauthorised use, reproduction or storage of the contents is expressly prohibited. If you have received this email in error, please delete it and any attachments immediately and advise the sender by return email or telephone. Deakin University does not warrant that this email and any attachments are error or virus free. From nirimo from jippii.fi Mon Apr 20 05:15:07 2009 From: nirimo from jippii.fi (nirimo@jippii.fi) Date: Mon Apr 20 09:30:07 2009 Subject: simply from single to double str DNA Message-ID: <4c31137a-3b3c-41c4-b726-3ee2809ce139@r33g2000yqn.googlegroups.com> Hei! I have a very simple question: anyone knows a free programme that would simply translate the copy-pasted single stranded DNA into double stranded DNA? Thank you :) NM From allison from nospam.com Mon Apr 20 09:44:29 2009 From: allison from nospam.com (Allison) Date: Mon Apr 20 10:42:34 2009 Subject: Separating GST from GST-fusion protein In-Reply-To: References: Message-ID: <49ec8633$0$9446$9a6e19ea@news.newshosting.com> DK wrote: > In article , "Paul J. Phelan" wrote: >> I think I asked this question before on this forum, but I thought I'd >> try it again just in case anyone has any new "tricks" or ideas, because >> I'm having the same old problem again. I am trying to purify a >> GST-fusion protein, and it's contaminated with GST. The GST-fusion has >> a mol. wt. of 40,000 and GST is 26,000, but on a Sephacryl S100 gel >> filtration column, both proteins are co-eluting in a single, beautiful >> peak. Anyone would think that such a textbook peak would only contain >> pure protein, but no - half of it is GST! I even re-ran the gel >> filtration column in a buffer containing 0.4 M NaCl to try to separate >> the two proteins, but I got the same single peak, so the GST must be >> still there. >> Now I am not talking about digesting a GST-fusion and then removing >> GST; I have no problem doing that, but I am trying to purify some >> intact GST-fusion, without taking GST with it: does anyone have a magic >> trick to get rid of GST? > > Because GST forms dimers (which is surprisingly little known and > responsible for probably countless incorrect conclusions in published > papers), you can only do it two ways: 1) cleave off GST if your > expression construct allows it, 2) run chromatography that > separates GSG from your GST fusion in the presence of high urea > concentration (at least 1M and as high as 2M may be necessary). > This is only an option of your fusion partner survives such > conditions. > > DK Getting rid of trace GST after cleavage is not trivial IMO. After expressing a GST fusion protein we cleaved away the GST using a thrombin cleavage site and ran an affinity column to get rid of uncleaved fusion protein and free GST. The resulting flowthrough has a single band on Coomassie blue stained gel but still has GST present as measured by ELISA. We've switched to plan B - a different construct. Allison From rdiazg from ipb.csic.es Mon Apr 20 10:14:47 2009 From: rdiazg from ipb.csic.es (=?ISO-8859-1?Q?Rosario_D=EDaz?=-=?ISO-8859-1?Q?Gonz=E1lez?=) Date: Mon Apr 20 10:42:40 2009 Subject: simply from single to double str DNA In-Reply-To: <4c31137a-3b3c-41c4-b726-3ee2809ce139@r33g2000yqn.googlegroups.com> References: <4c31137a-3b3c-41c4-b726-3ee2809ce139@r33g2000yqn.googlegroups.com> Message-ID: <20090420150600.M4558@ipb.csic.es> Hi! Lablife has an option for creating a new vector and work on it, I guess you could paste your sequence and get it ds. If it doesn't, try in this page: http://molbiol-tools.ca/molecular_biology_freeware.htm Hopefully some of them will work. Cheers! Rose. On Mon, 20 Apr 2009 03:15:07 -0700 (PDT), nirimo wrote > Hei! > > I have a very simple question: anyone knows a free programme that > would simply translate the copy-pasted single stranded DNA into > double stranded DNA? > > Thank you :) > > NM > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods Instituto de Parasitologia y Biomedicina "Lopez-Neyra" Avda. Conocimiento S/N Parque Tecnologico Ciencias de la Salud 18100 Armilla - GRANADA Spain Tlf: 958181621 Fax: 958181632 http://www.ipb.csic.es -- Open WebMail Project (http://openwebmail.org) From nick.theodorakis from gmail.com Mon Apr 20 10:46:23 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Mon Apr 20 14:08:33 2009 Subject: RNA extraction References: Message-ID: <4764e384-e403-41ed-9757-9a11ba9d7e83@f12g2000vbf.googlegroups.com> On Apr 20, 2:03?am, y...@deakin.edu.au wrote: > Hello, > > Just wondering if there is any way to extract RNA, prefer just the ? > rRNA, of e. coli without losing the proteins? Are you asking how to isolate ribosomes from E. coli? There are probably at least dozens of procedures one can find with a MEDLINE search, but most will probably have a centrifugation step in an ultracentrifuge; further clean up will depend in your specific needs. Can you give us a little more detail on what you are trying to do? Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From taliaferrod from mail.nih.gov Mon Apr 20 11:25:37 2009 From: taliaferrod from mail.nih.gov (Dwayne Taliaferro) Date: Mon Apr 20 14:08:38 2009 Subject: Separating GST from GST-fusion protein In-Reply-To: <49ec8633$0$9446$9a6e19ea@news.newshosting.com> Message-ID: This is also a problem with MBP protein tags as well. I had luck using a ion exchange columns and eluting using salt. > Getting rid of trace GST after cleavage is not trivial IMO. After > expressing a GST fusion protein we cleaved away the GST using a thrombin > cleavage site and ran an affinity column to get rid of uncleaved fusion > protein and free GST. The resulting flowthrough has a single band on > Coomassie blue stained gel but still has GST present as measured by ELISA. > We've switched to plan B - a different construct. > > Allison > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From engelbert_buxbaum from hotmail.com Mon Apr 20 11:18:22 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Apr 20 14:08:44 2009 Subject: Sodium Orthovanadate Preparation References: Message-ID: Am 18.04.2009, 15:57 Uhr, schrieb Lionel Brooks 3rd : > Sodium orthovanadate preparation: > This needs to be done under the fume hood > ? Prepare a 100 mM solution in double distilled water > ? Set pH to 9.0 with HCl > ? Boil until colorless > ? Cool to room temperature > ? Set pH to 9.0 again > ? Boil again until colorless > ? Repeat this cycle until the solution remains at pH 9.0 after boiling > and cooling > ? Bring up to the initial volume with water > ? Store in aliquots at -20 ?C > > Much to my chagrin the solution is not colorless after more than 30 min > of boiling. Any ideas on what I am doing wrong? There seems to be an impurity. Adding a spatula of activated charcoal before the first boiling and filtering afterwards might help. There may also be a trace of phosphate present, wich gives a yellow tint (or precipitate, if the concentration is high enough). Make sure that you have pure reagents and that the glassware is free of phosphate (detergent!) From bmacgreg from unc.edu Mon Apr 20 14:46:49 2009 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Mon Apr 20 15:20:17 2009 Subject: RNA extraction In-Reply-To: <200904201704.n3KH4Gh12954@net.bio.net> References: <200904201704.n3KH4Gh12954@net.bio.net> Message-ID: <616E14E5-2BE1-4595-87F7-C085DDDE7097@unc.edu> Hello Yvonne, TRI Reagent (or homemade substitute) might work for you - I think Applied Biosystems sells it now. That can separate RNA, DNA, and protein. Barbara On Apr 20, 2009, at 1:04 PM, methods-request@oat.bio.indiana.edu wrote: > > > Hello, > > Just wondering if there is any way to extract RNA, prefer just the > rRNA, of e. coli without losing the proteins? > > Yvonne > > Yvonne Fazendin, PhD Candidate > Deakin University, Pigdons Rd Geelong Victoria 3217 Australia. > Phone: 03 5227 2758 International: +61 3 5227 2758 > Email: ykfa@deakin.edu.au > Website: http://www.deakin.edu.au > Deakin University CRICOS Provider Code 00113B (Vic) > From itabajara.vaz from ufrgs.br Mon Apr 20 17:11:59 2009 From: itabajara.vaz from ufrgs.br (Itabajara Vaz - UFRGS) Date: Mon Apr 20 19:19:29 2009 Subject: bacterias 41(DE3) e C43(DE3) Message-ID: <49ECF32F.3090503@ufrgs.br> Hi, I am needing the C41(DE3), C41(DE3)pLysS and C43(DE3), C43(DE3)pLysS (E. coli bacterias) Does anybody have these bacterias? //Can anybody help me? Thank you very much, **C41(DE3), C41(DE3)pLysS and C43(DE3), C43(DE3)pLysS E. coli BL21(DE3) strains, like Lucigen's E. cloni EXPRESS Competent Cells provide reliable expression of many proteins cloned into T7 expression vectors (e.g., pET or Lucigen's pSMART?-cDNA vectors). However, in some cases expression is minimal or not detectable because the recombinant protein, when expressed, is deleterious or lethal to these standard BL21 strains. Examples of such toxic proteins include many membrane proteins, some cytoplasmic proteins, and nucleases. Unfortunately, successful expression of one or more toxic proteins is often important to the experimental goal. http://www.lucigen.com/catalog/index.php?cPath=47_58 sincerely -- Itabajara da Silva Vaz Junior Centro de Biotecnologia - UFRGS C.P. 15005 Av. Bento Goncalves 9500 Predio 43421 - Campus do Vale 91501-970 Porto Alegre RS Brasil Fone (51) 33086078 Fax (51)3308-73-09 [00 55 51 3308 7309] http://www.ufrgs.br/depbiot/201/cbt201.htm http://www.ufrgs.br/favet/imunovet From ebs15242 from creighton.edu Tue Apr 21 08:38:04 2009 From: ebs15242 from creighton.edu (Ed Siefker) Date: Tue Apr 21 12:17:45 2009 Subject: phenol in alcohol precip Message-ID: <49EDCC3C.2000906@creighton.edu> Hi. I was a little hasty in doing some phenol/chloroform preps of genomic DNA and apparently didn't remove all the phenol before adding ethanol to precipitate. How can I recover from this? I considered extracting with chloroform again, but I don't know if the phases will still separate with ethanol in there. Also, the DNA isn't in solution, so I'm guessing it would collect at the interface. If there were more DNA there, I'd just fish it out with a pipette tip, but it's a small prep. Any thoughts? Thanks a bunch -Ed From oliver.starks from gmail.com Tue Apr 21 09:26:50 2009 From: oliver.starks from gmail.com (oliver.starks@gmail.com) Date: Tue Apr 21 12:17:53 2009 Subject: Blue/white with pET15b, Rosetta2(DE3)? Message-ID: I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). Colonies appeared in appropriate numbers on an Amp/Cam plate, indicating the cells must have the vector. Without thinking it out I re-streaked a couple colonies onto another plate with IPTG/Xgal to re- test insert presence (I'm fairly confident in my original ligation but it's always possible that the cells from which I prepped my plasmid were cotransformed with empty pET15b as well). These produced blue coloration. But after thinking about it more, it seems like either blue should never be produced (regardless of insert presence/absence) or should always be produced (regardless of insert presence/absence) - because the insert is not disrupting a lacZ gene like it is in pBluescript or something of that nature. So...how's the blue being produced, and is it even evidence of my insert not being present? I'll be testing these with new preps and PCR or digest today or tomorrow anyway but thanks in advance for your help. From taliaferroD from mail.nih.gov Tue Apr 21 12:20:43 2009 From: taliaferroD from mail.nih.gov (Dwayne Taliaferro) Date: Tue Apr 21 15:46:54 2009 Subject: Genomic DNA isolation from mammalian cells In-Reply-To: <49EDCC3C.2000906@creighton.edu> Message-ID: Does anyone have a quick and reliable protocol for isolating gDNA from mammalian cells? I would be interested. From novalidaddress from nurfuerspam.de Tue Apr 21 16:09:10 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Apr 21 18:34:39 2009 Subject: phenol in alcohol precip References: Message-ID: Dear Ed, don't worry. Just rinse your pellet a few times with EtOH (and spin down shortly to make sure you don't loose the pellet).. If you plan downstream processing with enzymes, make sure all phenol has been removed. You might dare to check thesmell; your nose should turn out to be a very sensitive GC system with a neuronal detector attached to. Chloroform might not be that good choice, even that DNA doesn't dissolve in it, (regardless if there is EtOH and or phenol present), as it does not dissolve phenol as well as ethanol does. You might remember: the DNA (and other water soluble stuff) is in the phenol phase, lipids are in the chloroform phase and proteins are inbetween. Have fun! Wo From shifalich from rediffmail.com Tue Apr 21 21:45:33 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Tue Apr 21 22:10:42 2009 Subject: Expression and -N end rule Message-ID: <20090422024533.56211.qmail@f4mail204.rediffmail.com> Dear all! I had mentioned my problem earlier also in the forum but nobody replied. I am sure you great minds should have a solution to my problem. Please help me if you can! I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5 disulphide bonds.Mature protein has Arg as first residue after signal sequnece is clipped off. First of all, i tried yeast expression system. I expressed my protein in pPICZ alpha, under alpha factor signal sequence followed by Kex2 and Ste13 signal sequences, such that the mature protein will have native -N terminus with Arg as first residue, as of my protein. BUt, I could not see any protein expression even under different medium and proper aeration and whatever possible troubleshooting one could do. Recently, i read -N end rule which states that Arg is destabilizing residues and directs the protein for degradation to 26S proteasome. I checked with the company, they say, It hold true for every expression system. But, my question is, if that is the case, how the protein is stable in snake's venom. I have found the protein in the crude venom of the snake. Also, the protein is expressed under alpha factor signal sequence which is supposed to be secreted into the medium. Therefore,once the protein is out into medium, it is out of cell now, Kex2 and Ste13 cleavages should be happening outside the cell, therefore, there should be no proteasomal degradation. Am I right? After clipping off signal sequence, protein will be exported out, still will there be any proteasomal targetting of proteins with destabilizing residue at -N terminus? I read in -N end rule that, protein's life depends upon penultimate residue to Met. We know, for a protein to be synthesised, we need to have a start codon,i.e. Met. In -N end rule they say, Met will be removed be Metaminopeptidase(MAP), so if we express a protein we should not be counting Met residue in the expected MW? Because, not all mature protein sequences start from Met. Then,I tried expressing my protein using E. coli epression system, cDNA cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation). Met and Gly had to be added at -N terminus to maintain the reading frame. Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from ex-pression hosts. DNA is there but no protein. Please answer any, if not all, of the above question. i am struggling with all this for alst 8 months. Your suggestions and help will be highly appreciable. Thanks in advance. Shifali Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From novalidaddress from nurfuerspam.de Wed Apr 22 03:13:52 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Apr 22 07:48:47 2009 Subject: Expression and -N end rule References: Message-ID: <5db5bd9e-8539-425c-8f06-5da81a395164@m19g2000yqk.googlegroups.com> Hi Shifali, maybe your protein is toxic? you could change codon usage and/or try higher eucaryotic cells. To narrow down the effect of signal sequences etc.: Have you considered putting your protein (i.e its cDNA) and/or part of it in front of a reporter gene like GFP or insert just anything known in order to disrupt a possile action? Best regards, Wo From taliaferroD from mail.nih.gov Wed Apr 22 10:03:41 2009 From: taliaferroD from mail.nih.gov (Dwayne Taliaferro) Date: Wed Apr 22 11:37:33 2009 Subject: Genomic DNA isolation from mammalian cells (clarification) In-Reply-To: <8zsHl.69302$ua7.56187@newsfe17.iad> Message-ID: I am looking for a protocol that will allow me to purify genomic DNA from mammalian cells with the purpose of using it for Southern blotting and PCR. DT From bmacgreg from unc.edu Wed Apr 22 12:38:23 2009 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Wed Apr 22 13:43:57 2009 Subject: Phenol in alcohol precip In-Reply-To: <200904221704.n3MH4ih19378@net.bio.net> References: <200904221704.n3MH4ih19378@net.bio.net> Message-ID: <57265651-BCA9-4AEF-9FB2-3589C51BF6A9@unc.edu> Hi Ed, As long as your DNA is precipitated, you can just spin it down; take off the supernatant; resuspend the DNA in water (or your choice of buffer); and then repeat the chloroform extraction, if you're worried there's still phenol there. You should get MOST of the DNA back. Barbara > > > > From: Ed Siefker > Date: April 21, 2009 9:38:04 AM EDT > To: methods@magpie.bio.indiana.edu > Subject: phenol in alcohol precip > > > Hi. I was a little hasty in doing some phenol/chloroform > preps of genomic DNA and apparently didn't remove all > the phenol before adding ethanol to precipitate. How can > I recover from this? > > I considered extracting with chloroform again, but I don't > know if the phases will still separate with ethanol in there. > Also, the DNA isn't in solution, so I'm guessing it would > collect at the interface. If there were more DNA there, > I'd just fish it out with a pipette tip, but it's a small prep. > > Any thoughts? Thanks a bunch > -Ed > From pdeitik from bcm.tmc.edu Wed Apr 22 12:47:52 2009 From: pdeitik from bcm.tmc.edu (Deitiker, Philip R) Date: Wed Apr 22 13:44:03 2009 Subject: Genomic DNA isolation from mammalian cells In-Reply-To: References: <49EDCC3C.2000906@creighton.edu> Message-ID: >From Whole Blood, Qiagen Gentra PureGene DNA Isolation Kit. This will get Genomic DNA. One precaution on its use, fresh RBCs are hard to lyze, it requires washing in RBC lysis buffer and carefully drawing off the RBC layer from the White blood cells. Small levels of RBC contamination at the point of DNA extraction (White blood cell lysis and solubilization) can result in Fe/Heme that can really screw up the DNA prep. Blood should be collected in Acid Citrate Dextrose(ACD, BD Yellow Top Tubes) and 8 mls is more than enough for most efforts. For blood the kit is reliable. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Dwayne Taliaferro Sent: Tuesday, April 21, 2009 9:51 PM To: methods@magpie.bio.indiana.edu Subject: Genomic DNA isolation from mammalian cells Does anyone have a quick and reliable protocol for isolating gDNA from mammalian cells? I would be interested. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From kumar from lifesensors.com Wed Apr 22 14:40:45 2009 From: kumar from lifesensors.com (Suresh Kumar) Date: Wed Apr 22 15:30:41 2009 Subject: Expression and -N end rule In-Reply-To: <200904221704.n3MH4th19404@net.bio.net> References: <200904221704.n3MH4th19404@net.bio.net> Message-ID: <899CFF650DAF1245801B4A694F0E80646378FD@ls-sbs.ls.local> Hi Shifali, Your protein is stable in the snake venom, because the venom itself might not have the 26S proteasomal activity. I think venom proteins are secreted using secretion granules which shield them from the cellular 26S proteasomal activity. Regarding your troubles with expression, you could try these options. 1- Codon optimization. For a small protein such as yours the entire gene could be synthetically made with codon optimization for E.coli/Yeast/Insect with affordable price (at least here in the US). 2-SUMO expression system- Look into the SUMO expression system. http://lifesensors.com/www/sumo-expression-systems-c-55.html In this system, SUMO (Small Ubiquitin-like Modifier) tag is added to the N-terminus of your protein. This will prevent N-end rule degradation if there is any. Once the protein is expressed you can purify it using standard Ni-Affinity Purification (There is a His tag N-terminus to SUMO). SUMO tag is then cleaved with a SUMO specific protease which will leave the original N-terminus of your protein intact (Arginine in your case). This system offers vectors for E.coli, Yeast and Insect Cells. 3- Once codon optimized, you can try several standard conditions to optimize the expression. -Medium -IPTG (Low to High) -Temperature (18oC to 37oC) -Time -Lysis buffers -Protease/Proteasome inhibitors Hope this helps and best of luck! Suresh Disclaimer: I do not work for Life Sensors Inc., but my company has close ties with them and I have used the SUMO expression system successfully in E.coli. __________________________ This e-mail is from Progenra Inc. The e-mail and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. Any unauthorized dissemination or copying of this e-mail or its attachments, and any use or disclosure of any information contained in them, is strictly prohibited and may be illegal. If you have received this e-mail in error, please notify or telephone 610-644-6974 and delete it from your system -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of methods-request@oat.bio.indiana.edu Sent: Wednesday, April 22, 2009 1:05 PM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 47, Issue 16 Send Methods mailing list submissions to methods@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to methods-request@net.bio.net You can reach the person managing the list at methods-owner@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." Today's Topics: 1. phenol in alcohol precip (Ed Siefker) 2. Blue/white with pET15b, Rosetta2(DE3)? (oliver.starks@gmail.com) 3. Genomic DNA isolation from mammalian cells (Dwayne Taliaferro) 4. Re: Blue/white with pET15b, Rosetta2(DE3)? (DK) 5. Re: phenol in alcohol precip (WS) 6. Re: Genomic DNA isolation from mammalian cells (DK) 7. Expression and -N end rule (shifali chatrath) 8. Re: Expression and -N end rule (WS) 9. Genomic DNA isolation from mammalian cells (clarification) (Dwayne Taliaferro) ---------------------------------------------------------------------- Message: 1 Date: Tue, 21 Apr 2009 08:38:04 -0500 From: Ed Siefker Subject: phenol in alcohol precip To: methods@magpie.bio.indiana.edu Message-ID: <49EDCC3C.2000906@creighton.edu> Content-Type: text/plain; charset=ISO-8859-1; format=flowed Hi. I was a little hasty in doing some phenol/chloroform preps of genomic DNA and apparently didn't remove all the phenol before adding ethanol to precipitate. How can I recover from this? I considered extracting with chloroform again, but I don't know if the phases will still separate with ethanol in there. Also, the DNA isn't in solution, so I'm guessing it would collect at the interface. If there were more DNA there, I'd just fish it out with a pipette tip, but it's a small prep. Any thoughts? Thanks a bunch -Ed ------------------------------ Message: 2 Date: Tue, 21 Apr 2009 07:26:50 -0700 (PDT) From: oliver.starks@gmail.com Subject: Blue/white with pET15b, Rosetta2(DE3)? To: methods@net.bio.net Message-ID: Content-Type: text/plain; charset=ISO-8859-1 I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). Colonies appeared in appropriate numbers on an Amp/Cam plate, indicating the cells must have the vector. Without thinking it out I re-streaked a couple colonies onto another plate with IPTG/Xgal to re- test insert presence (I'm fairly confident in my original ligation but it's always possible that the cells from which I prepped my plasmid were cotransformed with empty pET15b as well). These produced blue coloration. But after thinking about it more, it seems like either blue should never be produced (regardless of insert presence/absence) or should always be produced (regardless of insert presence/absence) - because the insert is not disrupting a lacZ gene like it is in pBluescript or something of that nature. So...how's the blue being produced, and is it even evidence of my insert not being present? I'll be testing these with new preps and PCR or digest today or tomorrow anyway but thanks in advance for your help. ------------------------------ Message: 3 Date: Tue, 21 Apr 2009 13:20:43 -0400 From: Dwayne Taliaferro Subject: Genomic DNA isolation from mammalian cells To: Message-ID: Content-Type: text/plain; charset="US-ASCII" Does anyone have a quick and reliable protocol for isolating gDNA from mammalian cells? I would be interested. ------------------------------ Message: 4 Date: Tue, 21 Apr 2009 22:59:52 GMT From: dk@no.email.thankstospam.net (DK) Subject: Re: Blue/white with pET15b, Rosetta2(DE3)? To: methods@net.bio.net Message-ID: In article , oliver.starks@gmail.com wrote: >I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). >Colonies appeared in appropriate numbers on an Amp/Cam plate, >indicating the cells must have the vector. Without thinking it out I >re-streaked a couple colonies onto another plate with IPTG/Xgal to re- >test insert presence (I'm fairly confident in my original ligation but >it's always possible that the cells from which I prepped my plasmid >were cotransformed with empty pET15b as well). These produced blue >coloration. But after thinking about it more, it seems like either >blue should never be produced (regardless of insert presence/absence) >or should always be produced (regardless of insert presence/absence) - >because the insert is not disrupting a lacZ gene like it is in >pBluescript or something of that nature. > >So...how's the blue being produced, and is it even evidence of my >insert not being present? Rosetta(DE3) is simply BL21(DE3) with pRARE2 plasmid. The strain has WT lacZ, so naturally it makes blue colonies. It has nothing to do with your plasmid or it having an insert. DK ------------------------------ Message: 5 Date: Tue, 21 Apr 2009 14:09:10 -0700 (PDT) From: WS Subject: Re: phenol in alcohol precip To: methods@net.bio.net Message-ID: Content-Type: text/plain; charset=ISO-8859-1 Dear Ed, don't worry. Just rinse your pellet a few times with EtOH (and spin down shortly to make sure you don't loose the pellet).. If you plan downstream processing with enzymes, make sure all phenol has been removed. You might dare to check thesmell; your nose should turn out to be a very sensitive GC system with a neuronal detector attached to. Chloroform might not be that good choice, even that DNA doesn't dissolve in it, (regardless if there is EtOH and or phenol present), as it does not dissolve phenol as well as ethanol does. You might remember: the DNA (and other water soluble stuff) is in the phenol phase, lipids are in the chloroform phase and proteins are inbetween. Have fun! Wo ------------------------------ Message: 6 Date: Tue, 21 Apr 2009 23:22:51 GMT From: dk@no.email.thankstospam.net (DK) Subject: Re: Genomic DNA isolation from mammalian cells To: methods@net.bio.net Message-ID: <8zsHl.69302$ua7.56187@newsfe17.iad> In article , Dwayne Taliaferro wrote: >Does anyone have a quick and reliable protocol for isolating gDNA from >mammalian cells? I would be interested. The purpose of gDNA? For "high" yeild and reasonable purity, here is what I used on many different cells (dicty, insect, mammalian lines): Pellet cells from 10 ml culture, resuspend in 1% Triton X100, spin, keep pellet, lyse nuclei with 400 ul 1% SDS, 20 mM EDTA, add 600 ul chloroform, vortex 15", spin hard, take upper phase, repeat extraction 2X. Add RNAse to 0.1 mg/ml, let sit for 5', mix 3 volumes of Qiagen buffer GQ or 5 volumes of Qiagen buffer PB with one volume of chloroform-extracted material, load onto regular Qiagen miniprep column, wash 2X with PB, wash 2X with PE, elute with 100 ul of 1-10 mM Tris, pH 8.0-8.5 (or TE) heated to ~ 70C. DK ------------------------------ Message: 7 Date: 22 Apr 2009 02:45:33 -0000 From: "shifali chatrath" Subject: Expression and -N end rule To: Message-ID: <20090422024533.56211.qmail@f4mail204.rediffmail.com> Content-Type: text/plain; charset="ISO-8859-1" Dear all! I had mentioned my problem earlier also in the forum but nobody replied. I am sure you great minds should have a solution to my problem. Please help me if you can! I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5 disulphide bonds.Mature protein has Arg as first residue after signal sequnece is clipped off. First of all, i tried yeast expression system. I expressed my protein in pPICZ alpha, under alpha factor signal sequence followed by Kex2 and Ste13 signal sequences, such that the mature protein will have native -N terminus with Arg as first residue, as of my protein. BUt, I could not see any protein expression even under different medium and proper aeration and whatever possible troubleshooting one could do. Recently, i read -N end rule which states that Arg is destabilizing residues and directs the protein for degradation to 26S proteasome. I checked with the company, they say, It hold true for every expression system. But, my question is, if that is the case, how the protein is stable in snake's venom. I have found the protein in the crude venom of the snake. Also, the protein is expressed under alpha factor signal sequence which is supposed to be secreted into the medium. Therefore,once the protein is out into medium, it is out of cell now, Kex2 and Ste13 cleavages should be happening outside the cell, therefore, there should be no proteasomal degradation. Am I right? After clipping off signal sequence, protein will be exported out, still will there be any proteasomal targetting of proteins with destabilizing residue at -N terminus? I read in -N end rule that, protein's life depends upon penultimate residue to Met. We know, for a protein to be synthesised, we need to have a start codon,i.e. Met. In -N end rule they say, Met will be removed be Metaminopeptidase(MAP), so if we express a protein we should not be counting Met residue in the expected MW? Because, not all mature protein sequences start from Met. Then,I tried expressing my protein using E. coli epression system, cDNA cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation). Met and Gly had to be added at -N terminus to maintain the reading frame. Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from ex-pression hosts. DNA is there but no protein. Please answer any, if not all, of the above question. i am struggling with all this for alst 8 months. Your suggestions and help will be highly appreciable. Thanks in advance. Shifali Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 ------------------------------ Message: 8 Date: Wed, 22 Apr 2009 01:13:52 -0700 (PDT) From: WS Subject: Re: Expression and -N end rule To: methods@net.bio.net Message-ID: <5db5bd9e-8539-425c-8f06-5da81a395164@m19g2000yqk.googlegroups.com> Content-Type: text/plain; charset=ISO-8859-1 Hi Shifali, maybe your protein is toxic? you could change codon usage and/or try higher eucaryotic cells. To narrow down the effect of signal sequences etc.: Have you considered putting your protein (i.e its cDNA) and/or part of it in front of a reporter gene like GFP or insert just anything known in order to disrupt a possile action? Best regards, Wo ------------------------------ Message: 9 Date: Wed, 22 Apr 2009 11:03:41 -0400 From: Dwayne Taliaferro Subject: Genomic DNA isolation from mammalian cells (clarification) To: Message-ID: Content-Type: text/plain; charset="US-ASCII" I am looking for a protocol that will allow me to purify genomic DNA from mammalian cells with the purpose of using it for Southern blotting and PCR. DT ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 47, Issue 16 *************************************** From davidminde from gmail.com Wed Apr 22 17:10:53 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Wed Apr 22 18:58:30 2009 Subject: Expression and -N end rule In-Reply-To: <899CFF650DAF1245801B4A694F0E80646378FD@ls-sbs.ls.local> References: <200904221704.n3MH4th19404@net.bio.net> <899CFF650DAF1245801B4A694F0E80646378FD@ls-sbs.ls.local> Message-ID: <123243900904221510o64aa894bnad7d91afa3ec749b@mail.gmail.com> ... maybe, you even find a nearby colleague who is (as me) using a non-commercial SUMO system which does the exact same good job for overexpression and solubility in E. coli ;) SUMO system is really fun to use (if even more simple approaches like His-tag in a pET vector dont work). Great (3D) specificity, ultrafast cleavage and easy mass production of the corresponding enzyme for tag removal (Ulp1; we got 150 mg from 200 ml cells - enough for removing tag from some 37.5 g of fusion proteins which is probably more than the whole lab will need for the last couple years of my PhD ;) The literature on dramatic enhancements with codon optimized gene synthesis is very,very thin. So you better check if there are indeed extreme codons biases in your target before investing in that approach. On the other hand it might save labour cost in cloning work in your group and be cost-neutral for your small target. cheers, David Science is what happens while we are making other plans (~John Lennon) (PS I am currently working in the netherlands. Really a public sport here to save money :) From manishjnu8 from gmail.com Thu Apr 23 02:37:55 2009 From: manishjnu8 from gmail.com (manish mishra) Date: Thu Apr 23 10:20:37 2009 Subject: Methods Digest, Vol 47, Issue 16 In-Reply-To: <200904221704.n3MH4th19404@net.bio.net> References: <200904221704.n3MH4th19404@net.bio.net> Message-ID: Dear Wo, As you have written "You might > remember: the DNA (and other water soluble stuff) is in the phenol > phase, lipids are in the chloroform phase and proteins are inbetween." I am slightly confused. Is it really that DNA is in the phenolic phage? I think when we isolate genomic DNA by Phenol: Chloroform: Isoamyl alchohol , the pH is around 8. I think that nucleic acid should be present in aqueous phage at pH 8.The lower organic layer contains proteins dissolved by phenol and lipid dissolved by chloroform. I dont know at pH 8 whether RNA is in aqueous phage or at interface? At lower pH the RNA is in aquous phage and DNA is in phenolic phage. Am I correct. If possible kindly clear my confusion. with regards Manish On Wed, Apr 22, 2009 at 10:34 PM, wrote: > Send Methods mailing list submissions to > ? ? ? ?methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > ? ? ? ?http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > ? ? ? ?methods-request@net.bio.net > > You can reach the person managing the list at > ? ? ? ?methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > ? 1. phenol in alcohol precip (Ed Siefker) > ? 2. Blue/white with pET15b, Rosetta2(DE3)? (oliver.starks@gmail.com) > ? 3. Genomic DNA isolation from mammalian cells (Dwayne Taliaferro) > ? 4. Re: Blue/white with pET15b, Rosetta2(DE3)? (DK) > ? 5. Re: phenol in alcohol precip (WS) > ? 6. Re: Genomic DNA isolation from mammalian cells (DK) > ? 7. Expression and -N end rule (shifali ?chatrath) > ? 8. Re: Expression and -N end rule (WS) > ? 9. Genomic DNA isolation from mammalian cells (clarification) > ? ? ?(Dwayne Taliaferro) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 21 Apr 2009 08:38:04 -0500 > From: Ed Siefker > Subject: phenol in alcohol precip > To: methods@magpie.bio.indiana.edu > Message-ID: <49EDCC3C.2000906@creighton.edu> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Hi. ?I was a little hasty in doing some phenol/chloroform > preps of genomic DNA and apparently didn't remove all > the phenol before adding ethanol to precipitate. ?How can > I recover from this? > > I considered extracting with chloroform again, but I don't > know if the phases will still separate with ethanol in there. > Also, the DNA isn't in solution, so I'm guessing it would > collect at the interface. ?If there were more DNA there, > I'd just fish it out with a pipette tip, but it's a small prep. > > Any thoughts? ?Thanks a bunch > -Ed > > > > ------------------------------ > > Message: 2 > Date: Tue, 21 Apr 2009 07:26:50 -0700 (PDT) > From: oliver.starks@gmail.com > Subject: Blue/white with pET15b, Rosetta2(DE3)? > To: methods@net.bio.net > Message-ID: > ? ? ? ? > Content-Type: text/plain; charset=ISO-8859-1 > > I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). > Colonies appeared in appropriate numbers on an Amp/Cam plate, > indicating the cells must have the vector. ?Without thinking it out I > re-streaked a couple colonies onto another plate with IPTG/Xgal to re- > test insert presence (I'm fairly confident in my original ligation but > it's always possible that the cells from which I prepped my plasmid > were cotransformed with empty pET15b as well). ?These produced blue > coloration. ?But after thinking about it more, it seems like either > blue should never be produced (regardless of insert presence/absence) > or should always be produced (regardless of insert presence/absence) - > because the insert is not disrupting a lacZ gene like it is in > pBluescript or something of that nature. > > So...how's the blue being produced, and is it even evidence of my > insert not being present? > > I'll be testing these with new preps and PCR or digest today or > tomorrow anyway but thanks in advance for your help. > > > ------------------------------ > > Message: 3 > Date: Tue, 21 Apr 2009 13:20:43 -0400 > From: Dwayne Taliaferro > Subject: Genomic DNA isolation from mammalian cells > To: > Message-ID: > Content-Type: text/plain; ? ? ? charset="US-ASCII" > > Does anyone have a quick and reliable protocol for isolating gDNA from > mammalian cells? ?I ?would be interested. > > > > ------------------------------ > > Message: 4 > Date: Tue, 21 Apr 2009 22:59:52 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Blue/white with pET15b, Rosetta2(DE3)? > To: methods@net.bio.net > Message-ID: > > In article , oliver.starks@gmail.com wrote: >>I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). >>Colonies appeared in appropriate numbers on an Amp/Cam plate, >>indicating the cells must have the vector. ?Without thinking it out I >>re-streaked a couple colonies onto another plate with IPTG/Xgal to re- >>test insert presence (I'm fairly confident in my original ligation but >>it's always possible that the cells from which I prepped my plasmid >>were cotransformed with empty pET15b as well). ?These produced blue >>coloration. ?But after thinking about it more, it seems like either >>blue should never be produced (regardless of insert presence/absence) >>or should always be produced (regardless of insert presence/absence) - >>because the insert is not disrupting a lacZ gene like it is in >>pBluescript or something of that nature. >> >>So...how's the blue being produced, and is it even evidence of my >>insert not being present? > > Rosetta(DE3) is simply BL21(DE3) ?with pRARE2 plasmid. > The strain has WT lacZ, so naturally it makes blue colonies. > It has nothing to do with your plasmid or it having an insert. > > DK > > > ------------------------------ > > Message: 5 > Date: Tue, 21 Apr 2009 14:09:10 -0700 (PDT) > From: WS > Subject: Re: phenol in alcohol precip > To: methods@net.bio.net > Message-ID: > ? ? ? ? > Content-Type: text/plain; charset=ISO-8859-1 > > Dear Ed, > > don't worry. Just rinse your pellet a few times with EtOH (and spin > down shortly to make sure you don't loose the pellet).. If you plan > downstream processing with enzymes, make sure all phenol has been > removed. You might dare to check thesmell; your nose should turn out > to be a very sensitive GC system with a neuronal detector attached to. > Chloroform might not be that good choice, even that DNA doesn't > dissolve in it, (regardless if there is EtOH and or phenol present), > as it does not dissolve phenol as well as ethanol does. You might > remember: the DNA (and other water soluble stuff) is in the phenol > phase, lipids are in the chloroform phase and proteins are inbetween. > > Have fun! > > Wo > > > ------------------------------ > > Message: 6 > Date: Tue, 21 Apr 2009 23:22:51 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Genomic DNA isolation from mammalian cells > To: methods@net.bio.net > Message-ID: <8zsHl.69302$ua7.56187@newsfe17.iad> > > In article , Dwayne Taliaferro wrote: >>Does anyone have a quick and reliable protocol for isolating gDNA from >>mammalian cells? ?I ?would be interested. > > The purpose of gDNA? For "high" yeild and reasonable purity, > here is what I used on many different cells (dicty, insect, > mammalian lines): > > Pellet cells from 10 ml culture, resuspend in 1% Triton X100, > spin, keep pellet, lyse nuclei with 400 ul 1% SDS, 20 mM EDTA, > add 600 ul chloroform, vortex 15", spin hard, take upper phase, repeat > extraction 2X. Add RNAse to 0.1 mg/ml, let sit for 5', mix 3 volumes > of Qiagen buffer GQ or 5 volumes of Qiagen buffer PB with one > volume of chloroform-extracted material, load onto regular Qiagen > miniprep column, wash 2X with PB, wash 2X with PE, elute with > 100 ul of 1-10 mM Tris, pH 8.0-8.5 (or TE) heated to ~ 70C. > > DK > > > ------------------------------ > > Message: 7 > Date: 22 Apr 2009 02:45:33 -0000 > From: "shifali ?chatrath" > Subject: Expression and -N end rule > To: > Message-ID: <20090422024533.56211.qmail@f4mail204.rediffmail.com> > Content-Type: text/plain; charset="ISO-8859-1" > > Dear all! > I had mentioned my problem earlier also in the forum but nobody replied. I am sure you great minds should have a solution to my problem. Please help me if you can! > > I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5 disulphide bonds.Mature protein has Arg as first residue after signal sequnece is clipped off. > First of all, i tried yeast expression system. > I expressed my protein in pPICZ alpha, under alpha factor signal sequence followed by Kex2 and Ste13 signal sequences, such that the mature protein will have native -N terminus with Arg as first residue, as of my protein. BUt, I could not see any protein expression even under different medium and proper aeration and whatever possible troubleshooting one could do. > Recently, i read -N end rule which states that Arg is destabilizing residues and directs the protein for degradation to 26S proteasome. > I checked with the company, they say, It hold true for every expression system. > But, my question is, if that is the case, how the protein is stable in snake's venom. I have found the protein in the crude venom of the snake. > Also, the protein is expressed under alpha factor signal sequence which is supposed to be secreted into the medium. Therefore,once the protein is out into medium, it is out of cell now, Kex2 and Ste13 cleavages should be happening outside the cell, therefore, there should be no proteasomal degradation. Am I right? > > After clipping off signal sequence, protein will be exported out, still will there be any proteasomal targetting of proteins with destabilizing residue at -N terminus? > > I read in -N end rule that, protein's life depends upon penultimate residue to Met. We know, for a protein to be synthesised, we need to have ?a start codon,i.e. Met. In -N end rule they say, Met will be removed be Metaminopeptidase(MAP), so if we express a protein we should not be counting Met residue in the expected MW? Because, not all mature protein sequences start from Met. > > > Then,I tried expressing my protein using E. coli epression system, cDNA cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation). Met and Gly had to be added at -N terminus to maintain the reading frame. > Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from ex-pression hosts. DNA is there but no protein. > > Please answer any, if not all, of the above question. i am struggling with all this for alst 8 months. Your suggestions and help will be highly appreciable. > > Thanks in advance. > > Shifali > > > > Shifali Chatrath > Graduate Student > Protein science Lab > Dept. of Biological sciences > National University of Singapore > Singapore > +65-65161210 > +65-96393449 > > ------------------------------ > > Message: 8 > Date: Wed, 22 Apr 2009 01:13:52 -0700 (PDT) > From: WS > Subject: Re: Expression and -N end rule > To: methods@net.bio.net > Message-ID: > ? ? ? ?<5db5bd9e-8539-425c-8f06-5da81a395164@m19g2000yqk.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi Shifali, maybe your protein is toxic? you could change codon usage > and/or try higher eucaryotic cells. > > To narrow down the effect of signal sequences etc.: Have you > considered putting your protein (i.e its cDNA) and/or part of it in > front of a reporter gene like GFP or insert just anything known in > order to disrupt a possile action? > > Best regards, > > Wo > > > ------------------------------ > > Message: 9 > Date: Wed, 22 Apr 2009 11:03:41 -0400 > From: Dwayne Taliaferro > Subject: Genomic DNA isolation from mammalian cells (clarification) > To: > Message-ID: > Content-Type: text/plain; ? ? ? charset="US-ASCII" > > I am looking for a protocol that will allow me to purify genomic DNA from > mammalian cells with the purpose of using it for Southern blotting and PCR. > > DT > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 47, Issue 16 > *************************************** > -- Manish, Lab no 122, SBT , JNU. From novalidaddress from nurfuerspam.de Thu Apr 23 11:39:18 2009 From: novalidaddress from nurfuerspam.de (novalidaddress@nurfuerspam.de) Date: Thu Apr 23 12:00:49 2009 Subject: Methods Digest, Vol 47, Issue 16 In-Reply-To: References: <200904221704.n3MH4th19404@net.bio.net> Message-ID: <20090423163918.272740@gmx.net> Dear Manish, As the DNA is not in the chloroform phase, it is in the aqueous phase which contains the phenol (i.e. the phenolic phase), too. Maybe I haven't expressed that clearly enough (I wrote "phenol phase" which of course is not correct, there is no separate phenol phase, of course. Best regards, Wo -------- Original-Nachricht -------- > Datum: Thu, 23 Apr 2009 13:07:55 +0530 > Von: manish mishra > An: methods@oat.bio.indiana.edu > Betreff: Re: Methods Digest, Vol 47, Issue 16 > Dear Wo, > > As you have written > > "You might > > remember: the DNA (and other water soluble stuff) is in the phenol > > phase, lipids are in the chloroform phase and proteins are inbetween." > > I am slightly confused. Is it really that DNA is in the phenolic > phage? I think when we isolate genomic DNA by Phenol: Chloroform: > Isoamyl alchohol , the pH is around 8. > > I think that nucleic acid should be present in aqueous phage at pH > 8.The lower organic layer contains proteins dissolved by phenol and > lipid dissolved by chloroform. > I dont know at pH 8 whether RNA is in aqueous phage or at interface? > > At lower pH the RNA is in aquous phage and DNA is in phenolic phage. > > Am I correct. If possible kindly clear my confusion. > > with regards > Manish > -- Neu: GMX FreeDSL Komplettanschluss mit DSL 6.000 Flatrate + Telefonanschluss f?r nur 17,95 Euro/mtl.!* http://dslspecial.gmx.de/freedsl-surfflat/?ac=OM.AD.PD003K11308T4569a From SBrown from ccia.unsw.edu.au Thu Apr 23 12:17:33 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Thu Apr 23 13:58:20 2009 Subject: linearized vs uncut transfection References: <200904221704.n3MH4th19404@net.bio.net> Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A07D3B5B7@mail01.ccia.local> Hi guys, I would like to hear everyones thoughts on practical observations regarding transfection efficiency with regard to circular plasmid as opposed to linearising before transfection, i'm more interested on practical observations not so much on the theoretical implications. best regards scott Scott Brown PhD Candidate Molecular Carcinogenesis Program Children's Cancer Institute of Australia for Medical Research High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA Phone: +61 2 9382 1829 Fax: +61 2 9382 1850 Email: sbrown@ccia.unsw.edu.au Web: www.ccia.org.au Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error, please contact the sender immediately and destroy the original message. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu on behalf of manish mishra Sent: Thu 23/04/2009 5:37 PM To: methods@oat.bio.indiana.edu Subject: Re: Methods Digest, Vol 47, Issue 16 Dear Wo, As you have written "You might > remember: the DNA (and other water soluble stuff) is in the phenol > phase, lipids are in the chloroform phase and proteins are inbetween." I am slightly confused. Is it really that DNA is in the phenolic phage? I think when we isolate genomic DNA by Phenol: Chloroform: Isoamyl alchohol , the pH is around 8. I think that nucleic acid should be present in aqueous phage at pH 8.The lower organic layer contains proteins dissolved by phenol and lipid dissolved by chloroform. I dont know at pH 8 whether RNA is in aqueous phage or at interface? At lower pH the RNA is in aquous phage and DNA is in phenolic phage. Am I correct. If possible kindly clear my confusion. with regards Manish On Wed, Apr 22, 2009 at 10:34 PM, wrote: > Send Methods mailing list submissions to > ? ? ? ?methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > ? ? ? ?http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > ? ? ? ?methods-request@net.bio.net > > You can reach the person managing the list at > ? ? ? ?methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > ? 1. phenol in alcohol precip (Ed Siefker) > ? 2. Blue/white with pET15b, Rosetta2(DE3)? (oliver.starks@gmail.com) > ? 3. Genomic DNA isolation from mammalian cells (Dwayne Taliaferro) > ? 4. Re: Blue/white with pET15b, Rosetta2(DE3)? (DK) > ? 5. Re: phenol in alcohol precip (WS) > ? 6. Re: Genomic DNA isolation from mammalian cells (DK) > ? 7. Expression and -N end rule (shifali ?chatrath) > ? 8. Re: Expression and -N end rule (WS) > ? 9. Genomic DNA isolation from mammalian cells (clarification) > ? ? ?(Dwayne Taliaferro) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 21 Apr 2009 08:38:04 -0500 > From: Ed Siefker > Subject: phenol in alcohol precip > To: methods@magpie.bio.indiana.edu > Message-ID: <49EDCC3C.2000906@creighton.edu> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Hi. ?I was a little hasty in doing some phenol/chloroform > preps of genomic DNA and apparently didn't remove all > the phenol before adding ethanol to precipitate. ?How can > I recover from this? > > I considered extracting with chloroform again, but I don't > know if the phases will still separate with ethanol in there. > Also, the DNA isn't in solution, so I'm guessing it would > collect at the interface. ?If there were more DNA there, > I'd just fish it out with a pipette tip, but it's a small prep. > > Any thoughts? ?Thanks a bunch > -Ed > > > > ------------------------------ > > Message: 2 > Date: Tue, 21 Apr 2009 07:26:50 -0700 (PDT) > From: oliver.starks@gmail.com > Subject: Blue/white with pET15b, Rosetta2(DE3)? > To: methods@net.bio.net > Message-ID: > ? ? ? ? > Content-Type: text/plain; charset=ISO-8859-1 > > I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). > Colonies appeared in appropriate numbers on an Amp/Cam plate, > indicating the cells must have the vector. ?Without thinking it out I > re-streaked a couple colonies onto another plate with IPTG/Xgal to re- > test insert presence (I'm fairly confident in my original ligation but > it's always possible that the cells from which I prepped my plasmid > were cotransformed with empty pET15b as well). ?These produced blue > coloration. ?But after thinking about it more, it seems like either > blue should never be produced (regardless of insert presence/absence) > or should always be produced (regardless of insert presence/absence) - > because the insert is not disrupting a lacZ gene like it is in > pBluescript or something of that nature. > > So...how's the blue being produced, and is it even evidence of my > insert not being present? > > I'll be testing these with new preps and PCR or digest today or > tomorrow anyway but thanks in advance for your help. > > > ------------------------------ > > Message: 3 > Date: Tue, 21 Apr 2009 13:20:43 -0400 > From: Dwayne Taliaferro > Subject: Genomic DNA isolation from mammalian cells > To: > Message-ID: > Content-Type: text/plain; ? ? ? charset="US-ASCII" > > Does anyone have a quick and reliable protocol for isolating gDNA from > mammalian cells? ?I ?would be interested. > > > > ------------------------------ > > Message: 4 > Date: Tue, 21 Apr 2009 22:59:52 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Blue/white with pET15b, Rosetta2(DE3)? > To: methods@net.bio.net > Message-ID: > > In article , oliver.starks@gmail.com wrote: >>I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). >>Colonies appeared in appropriate numbers on an Amp/Cam plate, >>indicating the cells must have the vector. ?Without thinking it out I >>re-streaked a couple colonies onto another plate with IPTG/Xgal to re- >>test insert presence (I'm fairly confident in my original ligation but >>it's always possible that the cells from which I prepped my plasmid >>were cotransformed with empty pET15b as well). ?These produced blue >>coloration. ?But after thinking about it more, it seems like either >>blue should never be produced (regardless of insert presence/absence) >>or should always be produced (regardless of insert presence/absence) - >>because the insert is not disrupting a lacZ gene like it is in >>pBluescript or something of that nature. >> >>So...how's the blue being produced, and is it even evidence of my >>insert not being present? > > Rosetta(DE3) is simply BL21(DE3) ?with pRARE2 plasmid. > The strain has WT lacZ, so naturally it makes blue colonies. > It has nothing to do with your plasmid or it having an insert. > > DK > > > ------------------------------ > > Message: 5 > Date: Tue, 21 Apr 2009 14:09:10 -0700 (PDT) > From: WS > Subject: Re: phenol in alcohol precip > To: methods@net.bio.net > Message-ID: > ? ? ? ? > Content-Type: text/plain; charset=ISO-8859-1 > > Dear Ed, > > don't worry. Just rinse your pellet a few times with EtOH (and spin > down shortly to make sure you don't loose the pellet).. If you plan > downstream processing with enzymes, make sure all phenol has been > removed. You might dare to check thesmell; your nose should turn out > to be a very sensitive GC system with a neuronal detector attached to. > Chloroform might not be that good choice, even that DNA doesn't > dissolve in it, (regardless if there is EtOH and or phenol present), > as it does not dissolve phenol as well as ethanol does. You might > remember: the DNA (and other water soluble stuff) is in the phenol > phase, lipids are in the chloroform phase and proteins are inbetween. > > Have fun! > > Wo > > > ------------------------------ > > Message: 6 > Date: Tue, 21 Apr 2009 23:22:51 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Genomic DNA isolation from mammalian cells > To: methods@net.bio.net > Message-ID: <8zsHl.69302$ua7.56187@newsfe17.iad> > > In article , Dwayne Taliaferro wrote: >>Does anyone have a quick and reliable protocol for isolating gDNA from >>mammalian cells? ?I ?would be interested. > > The purpose of gDNA? For "high" yeild and reasonable purity, > here is what I used on many different cells (dicty, insect, > mammalian lines): > > Pellet cells from 10 ml culture, resuspend in 1% Triton X100, > spin, keep pellet, lyse nuclei with 400 ul 1% SDS, 20 mM EDTA, > add 600 ul chloroform, vortex 15", spin hard, take upper phase, repeat > extraction 2X. Add RNAse to 0.1 mg/ml, let sit for 5', mix 3 volumes > of Qiagen buffer GQ or 5 volumes of Qiagen buffer PB with one > volume of chloroform-extracted material, load onto regular Qiagen > miniprep column, wash 2X with PB, wash 2X with PE, elute with > 100 ul of 1-10 mM Tris, pH 8.0-8.5 (or TE) heated to ~ 70C. > > DK > > > ------------------------------ > > Message: 7 > Date: 22 Apr 2009 02:45:33 -0000 > From: "shifali ?chatrath" > Subject: Expression and -N end rule > To: > Message-ID: <20090422024533.56211.qmail@f4mail204.rediffmail.com> > Content-Type: text/plain; charset="ISO-8859-1" > > Dear all! > I had mentioned my problem earlier also in the forum but nobody replied. I am sure you great minds should have a solution to my problem. Please help me if you can! > > I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5 disulphide bonds.Mature protein has Arg as first residue after signal sequnece is clipped off. > First of all, i tried yeast expression system. > I expressed my protein in pPICZ alpha, under alpha factor signal sequence followed by Kex2 and Ste13 signal sequences, such that the mature protein will have native -N terminus with Arg as first residue, as of my protein. BUt, I could not see any protein expression even under different medium and proper aeration and whatever possible troubleshooting one could do. > Recently, i read -N end rule which states that Arg is destabilizing residues and directs the protein for degradation to 26S proteasome. > I checked with the company, they say, It hold true for every expression system. > But, my question is, if that is the case, how the protein is stable in snake's venom. I have found the protein in the crude venom of the snake. > Also, the protein is expressed under alpha factor signal sequence which is supposed to be secreted into the medium. Therefore,once the protein is out into medium, it is out of cell now, Kex2 and Ste13 cleavages should be happening outside the cell, therefore, there should be no proteasomal degradation. Am I right? > > After clipping off signal sequence, protein will be exported out, still will there be any proteasomal targetting of proteins with destabilizing residue at -N terminus? > > I read in -N end rule that, protein's life depends upon penultimate residue to Met. We know, for a protein to be synthesised, we need to have ?a start codon,i.e. Met. In -N end rule they say, Met will be removed be Metaminopeptidase(MAP), so if we express a protein we should not be counting Met residue in the expected MW? Because, not all mature protein sequences start from Met. > > > Then,I tried expressing my protein using E. coli epression system, cDNA cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any expression. Cells were growing normaly.(I hope, the protein is not inhibiting transcription and translation). Met and Gly had to be added at -N terminus to maintain the reading frame. > Protein is 10Kda and was cloned without tag sequence. I mean expected protein should be 10kDa.I confirmed the sequences before cloning and even plasmid isolated from ex-pression hosts. DNA is there but no protein. > > Please answer any, if not all, of the above question. i am struggling with all this for alst 8 months. Your suggestions and help will be highly appreciable. > > Thanks in advance. > > Shifali > > > > Shifali Chatrath > Graduate Student > Protein science Lab > Dept. of Biological sciences > National University of Singapore > Singapore > +65-65161210 > +65-96393449 > > ------------------------------ > > Message: 8 > Date: Wed, 22 Apr 2009 01:13:52 -0700 (PDT) > From: WS > Subject: Re: Expression and -N end rule > To: methods@net.bio.net > Message-ID: > ? ? ? ?<5db5bd9e-8539-425c-8f06-5da81a395164@m19g2000yqk.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi Shifali, maybe your protein is toxic? you could change codon usage > and/or try higher eucaryotic cells. > > To narrow down the effect of signal sequences etc.: Have you > considered putting your protein (i.e its cDNA) and/or part of it in > front of a reporter gene like GFP or insert just anything known in > order to disrupt a possile action? > > Best regards, > > Wo > > > ------------------------------ > > Message: 9 > Date: Wed, 22 Apr 2009 11:03:41 -0400 > From: Dwayne Taliaferro > Subject: Genomic DNA isolation from mammalian cells (clarification) > To: > Message-ID: > Content-Type: text/plain; ? ? ? charset="US-ASCII" > > I am looking for a protocol that will allow me to purify genomic DNA from > mammalian cells with the purpose of using it for Southern blotting and PCR. > > DT > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 47, Issue 16 > *************************************** > -- Manish, Lab no 122, SBT , JNU. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From tk from shaggy.csail.mit.edu Thu Apr 23 12:45:26 2009 From: tk from shaggy.csail.mit.edu (Tom Knight) Date: Thu Apr 23 13:58:30 2009 Subject: Methods Digest, Vol 47, Issue 16 References: <200904221704.n3MH4th19404@net.bio.net> Message-ID: novalidaddress@nurfuerspam.de writes: > As the DNA is not in the chloroform phase, it is in the aqueous > phase which contains the phenol (i.e. the phenolic phase), > too. Maybe I haven't expressed that clearly enough (I wrote "phenol > phase" which of course is not correct, there is no separate phenol > phase, of course. The majority of the phenol partitions along with the chloroform phase. Small amounts of phenol partition into the aqueous phase, along with the DNA. A subsequent extraction of the aqueous phase with chloroform is used to remove most of the remaining phenol from the aqueous phase, followed typically with ethanol preciptiation. From verokeim from yahoo.com Fri Apr 24 10:05:48 2009 From: verokeim from yahoo.com (Veronica Keim) Date: Fri Apr 24 13:28:18 2009 Subject: RNAi consulting Message-ID: <365536.13613.qm@web39505.mail.mud.yahoo.com> I?m working with an inducible interference RNA system in Arabidopsis thaliana,?which?is activated by the addition of 17 beta estradiol. I?ve read a few publications where mention?that this compound is supplied by Sigma Aldrich. My problem is that I wasn?t able to find the specific type of 17 beta estradiol that they use and?Sigma offer a lot of options. Does anybody know which kind of 17 beta estradiol should I use for this inducible system? Thanks ? Veronica Keim ? PhD Student Facultad de Farmacia Universidad de Barcelona Spain ____________________________________________________________________________________ ?Obt?n la mejor experiencia en la web! Descarga gratis el nuevo Internet Explorer 8. http://downloads.yahoo.com/ieak8/?l=e1 From shifalich from rediffmail.com Sat Apr 25 06:18:45 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat Apr 25 12:00:44 2009 Subject: Expression and -N end rule Message-ID: <1240432329.S.16756.20090.f4mail-234-208.rediffmail.com.old.1240658324.14003@webmail.rediffmail.com> Dear all! Thanks for the suggestions. SUMO was good one. Will try and get back to you after getting it done. Thanks again Shifai On Thu, 23 Apr 2009 02:02:09 +0530 wrote >Hi Shifali, > >Your protein is stable in the snake venom, because the venom itself >might not have the 26S proteasomal activity. I think venom proteins are >secreted using secretion granules which shield them from the cellular >26S proteasomal activity. > >Regarding your troubles with expression, you could try these options. > >1- Codon optimization. For a small protein such as yours the entire gene >could be synthetically made with codon optimization for >E.coli/Yeast/Insect with affordable price (at least here in the US). > >2-SUMO expression system- Look into the SUMO expression system. > >http://lifesensors.com/www/sumo-expression-systems-c-55.html > >In this system, SUMO (Small Ubiquitin-like Modifier) tag is added to the >N-terminus of your protein. This will prevent N-end rule degradation if >there is any. Once the protein is expressed you can purify it using >standard Ni-Affinity Purification (There is a His tag N-terminus to >SUMO). SUMO tag is then cleaved with a SUMO specific protease which will >leave the original N-terminus of your protein intact (Arginine in your >case). > >This system offers vectors for E.coli, Yeast and Insect Cells. > >3- Once codon optimized, you can try several standard conditions to >optimize the expression. > >-Medium >-IPTG (Low to High) >-Temperature (18oC to 37oC) >-Time >-Lysis buffers >-Protease/Proteasome inhibitors > >Hope this helps and best of luck! > >Suresh > >Disclaimer: I do not work for Life Sensors Inc., but my company has >close ties with them and I have used the SUMO expression system >successfully in E.coli. > >__________________________ >This e-mail is from Progenra Inc. The e-mail and any files transmitted >with it are confidential and intended solely for the use of the >individual or entity to whom they are addressed. Any unauthorized >dissemination or copying of this e-mail or its attachments, and any use >or disclosure of any information contained in them, is strictly >prohibited and may be illegal. If you have received this e-mail in >error, please notify or telephone 610-644-6974 and delete it from your >system >-----Original Message----- >From: methods-bounces@oat.bio.indiana.edu >[mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of >methods-request@oat.bio.indiana.edu >Sent: Wednesday, April 22, 2009 1:05 PM >To: methods@magpie.bio.indiana.edu >Subject: Methods Digest, Vol 47, Issue 16 > >Send Methods mailing list submissions to >???methods@net.bio.net > >To subscribe or unsubscribe via the World Wide Web, visit >???http://www.bio.net/biomail/listinfo/methods >or, via email, send a message with subject or body 'help' to >???methods-request@net.bio.net > >You can reach the person managing the list at >???methods-owner@net.bio.net > >When replying, please edit your Subject line so it is more specific >than "Re: Contents of Methods digest..." > > >Today's Topics: > > ? 1. phenol in alcohol precip (Ed Siefker) > ? 2. Blue/white with pET15b, Rosetta2(DE3)? (oliver.starks@gmail.com) > ? 3. Genomic DNA isolation from mammalian cells (Dwayne Taliaferro) > ? 4. Re: Blue/white with pET15b, Rosetta2(DE3)? (DK) > ? 5. Re: phenol in alcohol precip (WS) > ? 6. Re: Genomic DNA isolation from mammalian cells (DK) > ? 7. Expression and -N end rule (shifali ?chatrath) > ? 8. Re: Expression and -N end rule (WS) > ? 9. Genomic DNA isolation from mammalian cells (clarification) > ? ? ?(Dwayne Taliaferro) > > >---------------------------------------------------------------------- > >Message: 1 >Date: Tue, 21 Apr 2009 08:38:04 -0500 >From: Ed Siefker >Subject: phenol in alcohol precip >To: methods@magpie.bio.indiana.edu >Message-ID: >Content-Type: text/plain; charset=ISO-8859-1; format=flowed > >Hi. ?I was a little hasty in doing some phenol/chloroform >preps of genomic DNA and apparently didn't remove all >the phenol before adding ethanol to precipitate. ?How can >I recover from this? > >I considered extracting with chloroform again, but I don't >know if the phases will still separate with ethanol in there. >Also, the DNA isn't in solution, so I'm guessing it would >collect at the interface. ?If there were more DNA there, >I'd just fish it out with a pipette tip, but it's a small prep. > >Any thoughts? ?Thanks a bunch >-Ed > > > >------------------------------ > >Message: 2 >Date: Tue, 21 Apr 2009 07:26:50 -0700 (PDT) >From: oliver.starks@gmail.com >Subject: Blue/white with pET15b, Rosetta2(DE3)? >To: methods@net.bio.net >Message-ID: >??? > >Content-Type: text/plain; charset=ISO-8859-1 > >I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). >Colonies appeared in appropriate numbers on an Amp/Cam plate, >indicating the cells must have the vector. ?Without thinking it out I >re-streaked a couple colonies onto another plate with IPTG/Xgal to re- >test insert presence (I'm fairly confident in my original ligation but >it's always possible that the cells from which I prepped my plasmid >were cotransformed with empty pET15b as well). ?These produced blue >coloration. ?But after thinking about it more, it seems like either >blue should never be produced (regardless of insert presence/absence) >or should always be produced (regardless of insert presence/absence) - >because the insert is not disrupting a lacZ gene like it is in >pBluescript or something of that nature. > >So...how's the blue being produced, and is it even evidence of my >insert not being present? > >I'll be testing these with new preps and PCR or digest today or >tomorrow anyway but thanks in advance for your help. > > >------------------------------ > >Message: 3 >Date: Tue, 21 Apr 2009 13:20:43 -0400 >From: Dwayne Taliaferro >Subject: Genomic DNA isolation from mammalian cells >To: >Message-ID: >Content-Type: text/plain;???charset="US-ASCII" > >Does anyone have a quick and reliable protocol for isolating gDNA from >mammalian cells? ?I ?would be interested. > > > >------------------------------ > >Message: 4 >Date: Tue, 21 Apr 2009 22:59:52 GMT >From: dk@no.email.thankstospam.net (DK) >Subject: Re: Blue/white with pET15b, Rosetta2(DE3)? >To: methods@net.bio.net >Message-ID: > >In article >, >oliver.starks@gmail.com wrote: >>I've got my gene inserted in pET15b, and I transformed Rosetta2(DE3). >>Colonies appeared in appropriate numbers on an Amp/Cam plate, >>indicating the cells must have the vector. ?Without thinking it out I >>re-streaked a couple colonies onto another plate with IPTG/Xgal to re- >>test insert presence (I'm fairly confident in my original ligation but >>it's always possible that the cells from which I prepped my plasmid >>were cotransformed with empty pET15b as well). ?These produced blue >>coloration. ?But after thinking about it more, it seems like either >>blue should never be produced (regardless of insert presence/absence) >>or should always be produced (regardless of insert presence/absence) - >>because the insert is not disrupting a lacZ gene like it is in >>pBluescript or something of that nature. >> >>So...how's the blue being produced, and is it even evidence of my >>insert not being present? > >Rosetta(DE3) is simply BL21(DE3) ?with pRARE2 plasmid. >The strain has WT lacZ, so naturally it makes blue colonies. >It has nothing to do with your plasmid or it having an insert. > >DK > > >------------------------------ > >Message: 5 >Date: Tue, 21 Apr 2009 14:09:10 -0700 (PDT) >From: WS >Subject: Re: phenol in alcohol precip >To: methods@net.bio.net >Message-ID: >??? > >Content-Type: text/plain; charset=ISO-8859-1 > >Dear Ed, > >don't worry. Just rinse your pellet a few times with EtOH (and spin >down shortly to make sure you don't loose the pellet).. If you plan >downstream processing with enzymes, make sure all phenol has been >removed. You might dare to check thesmell; your nose should turn out >to be a very sensitive GC system with a neuronal detector attached to. >Chloroform might not be that good choice, even that DNA doesn't >dissolve in it, (regardless if there is EtOH and or phenol present), >as it does not dissolve phenol as well as ethanol does. You might >remember: the DNA (and other water soluble stuff) is in the phenol >phase, lipids are in the chloroform phase and proteins are inbetween. > >Have fun! > >Wo > > >------------------------------ > >Message: 6 >Date: Tue, 21 Apr 2009 23:22:51 GMT >From: dk@no.email.thankstospam.net (DK) >Subject: Re: Genomic DNA isolation from mammalian cells >To: methods@net.bio.net >Message-ID: > >In article , Dwayne >Taliaferro wrote: >>Does anyone have a quick and reliable protocol for isolating gDNA from >>mammalian cells? ?I ?would be interested. > >The purpose of gDNA? For "high" yeild and reasonable purity, >here is what I used on many different cells (dicty, insect, >mammalian lines): > >Pellet cells from 10 ml culture, resuspend in 1% Triton X100, >spin, keep pellet, lyse nuclei with 400 ul 1% SDS, 20 mM EDTA, >add 600 ul chloroform, vortex 15", spin hard, take upper phase, repeat >extraction 2X. Add RNAse to 0.1 mg/ml, let sit for 5', mix 3 volumes >of Qiagen buffer GQ or 5 volumes of Qiagen buffer PB with one >volume of chloroform-extracted material, load onto regular Qiagen >miniprep column, wash 2X with PB, wash 2X with PE, elute with >100 ul of 1-10 mM Tris, pH 8.0-8.5 (or TE) heated to ~ 70C. > >DK > > >------------------------------ > >Message: 7 >Date: 22 Apr 2009 02:45:33 -0000 >From: "shifali ?chatrath" >Subject: Expression and -N end rule >To: >Message-ID: >Content-Type: text/plain; charset="ISO-8859-1" > >Dear all! >I had mentioned my problem earlier also in the forum but nobody replied. >I am sure you great minds should have a solution to my problem. Please >help me if you can! > >I want to express a snake venom protein,~ 10kDa, 10 cys residues and 5 >disulphide bonds.Mature protein has Arg as first residue after signal >sequnece is clipped off. >First of all, i tried yeast expression system. >I expressed my protein in pPICZ alpha, under alpha factor signal >sequence followed by Kex2 and Ste13 signal sequences, such that the >mature protein will have native -N terminus with Arg as first residue, >as of my protein. BUt, I could not see any protein expression even under >different medium and proper aeration and whatever possible >troubleshooting one could do. >Recently, i read -N end rule which states that Arg is destabilizing >residues and directs the protein for degradation to 26S proteasome. >I checked with the company, they say, It hold true for every expression >system. >But, my question is, if that is the case, how the protein is stable in >snake's venom. I have found the protein in the crude venom of the snake. >Also, the protein is expressed under alpha factor signal sequence which >is supposed to be secreted into the medium. Therefore,once the protein >is out into medium, it is out of cell now, Kex2 and Ste13 cleavages >should be happening outside the cell, therefore, there should be no >proteasomal degradation. Am I right? > >After clipping off signal sequence, protein will be exported out, still >will there be any proteasomal targetting of proteins with destabilizing >residue at -N terminus? > >I read in -N end rule that, protein's life depends upon penultimate >residue to Met. We know, for a protein to be synthesised, we need to >have ?a start codon,i.e. Met. In -N end rule they say, Met will be >removed be Metaminopeptidase(MAP), so if we express a protein we should >not be counting Met residue in the expected MW? Because, not all mature >protein sequences start from Met. > > >Then,I tried expressing my protein using E. coli epression system, cDNA >cloned in pET19b in Rosetta gami cells and RIL cells.I couldn't see any >expression. Cells were growing normaly.(I hope, the protein is not >inhibiting transcription and translation). Met and Gly had to be added >at -N terminus to maintain the reading frame. >Protein is 10Kda and was cloned without tag sequence. I mean expected >protein should be 10kDa.I confirmed the sequences before cloning and >even plasmid isolated from ex-pression hosts. DNA is there but no >protein. > >Please answer any, if not all, of the above question. i am struggling >with all this for alst 8 months. Your suggestions and help will be >highly appreciable. > >Thanks in advance. > >Shifali > > > >Shifali Chatrath >Graduate Student >Protein science Lab >Dept. of Biological sciences >National University of Singapore >Singapore >+65-65161210 >+65-96393449 > >------------------------------ > >Message: 8 >Date: Wed, 22 Apr 2009 01:13:52 -0700 (PDT) >From: WS >Subject: Re: Expression and -N end rule >To: methods@net.bio.net >Message-ID: >??? > >Content-Type: text/plain; charset=ISO-8859-1 > >Hi Shifali, maybe your protein is toxic? you could change codon usage >and/or try higher eucaryotic cells. > >To narrow down the effect of signal sequences etc.: Have you >considered putting your protein (i.e its cDNA) and/or part of it in >front of a reporter gene like GFP or insert just anything known in >order to disrupt a possile action? > >Best regards, > >Wo > > >------------------------------ > >Message: 9 >Date: Wed, 22 Apr 2009 11:03:41 -0400 >From: Dwayne Taliaferro >Subject: Genomic DNA isolation from mammalian cells (clarification) >To: >Message-ID: >Content-Type: text/plain;???charset="US-ASCII" > >I am looking for a protocol that will allow me to purify genomic DNA >from >mammalian cells with the purpose of using it for Southern blotting and >PCR. > >DT > > > >------------------------------ > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods > >End of Methods Digest, Vol 47, Issue 16 >*************************************** > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods > Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From cjtcdy from gmail.com Sat Apr 25 13:51:09 2009 From: cjtcdy from gmail.com (chiranjit chowdhury) Date: Sun Apr 26 11:45:52 2009 Subject: EDTA for lysozyme action Message-ID: <2021705c0904251151j13b0dd5axc1a946da0f405760@mail.gmail.com> Can any one tell why EDTA is required for lysozyme action specially in case of G- bacteria? What is the mechanism behind it? Regards -- Chiranjit Chowdhury Department of Biotechnology Indian Institute of Technology, Kharagpur Kharagpur, West Bengal, India Pin: 721302 Official email: chiranjit.chowdhury@iitkgp.ac.in From davidminde from gmail.com Sun Apr 26 09:12:29 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Sun Apr 26 11:46:08 2009 Subject: Expression and -N end rule In-Reply-To: <1240432329.S.16756.20090.f4mail-234-208.rediffmail.com.old.1240658324.14003@webmail.rediffmail.com> References: <1240432329.S.16756.20090.f4mail-234-208.rediffmail.com.old.1240658324.14003@webmail.rediffmail.com> Message-ID: <123243900904260712p7cb03d92x8c4aaaa473c38436@mail.gmail.com> y, and SUMO is a quite small tag. ... Can also be easlily implemented in basically any (expression) vector easily via RF cloning (s.attached .. which really works nicely if you have competent cells as required for good quickchange for just simple pointmutations as well and use e.g. Phusion as good polymerase) or of course the other way round if you are not dealing with giants like tition (with some 33 000 aa which is not very convenient for PCR :). best, David From alejandro.martin from cigb.edu.cu Wed Apr 29 07:26:57 2009 From: alejandro.martin from cigb.edu.cu (Alejandro Miguel Martin Dunn) Date: Wed Apr 29 10:09:33 2009 Subject: Expression and -N end rule In-Reply-To: <20090422024533.56211.qmail@f4mail204.rediffmail.com> References: <20090422024533.56211.qmail@f4mail204.rediffmail.com> Message-ID: <00925AA44B42854BB6C9BE98C589E86A013B4799@frodo.cigb.edu.cu> Shifali, the N-end rule works at the cytoplasm; it has no influence on the half-life of proteins secreted into the ER (as should be the case for your venom protein in Pichia). Also (at least in E. coli) remember that the amino-terminal methionine is not always removed by MAP; this depends on the bulkiness of the side chain for the second a.a. Unsurprisingly, residues which are destabilizing according to the N-end rule often inhibit removal of the N-terminal Met by MAP when they are in the second position. how did you check for expression in yeast? did you also test cell lysates? Straight SDS-PAGE or Western blotting? > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu > [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of > shifali chatrath > Sent: Tuesday, April 21, 2009 10:46 PM > To: methods@magpie.bio.indiana.edu > Subject: Expression and -N end rule > > Dear all! > I had mentioned my problem earlier also in the forum but > nobody replied. I am sure you great minds should have a > solution to my problem. Please help me if you can! > > I want to express a snake venom protein,~ 10kDa, 10 cys > residues and 5 disulphide bonds.Mature protein has Arg as > first residue after signal sequnece is clipped off. > First of all, i tried yeast expression system. > I expressed my protein in pPICZ alpha, under alpha factor > signal sequence followed by Kex2 and Ste13 signal sequences, > such that the mature protein will have native -N terminus > with Arg as first residue, as of my protein. BUt, I could not > see any protein expression even under different medium and > proper aeration and whatever possible troubleshooting one could do. > Recently, i read -N end rule which states that Arg is > destabilizing residues and directs the protein for > degradation to 26S proteasome. > I checked with the company, they say, It hold true for every > expression system. > But, my question is, if that is the case, how the protein is > stable in snake's venom. I have found the protein in the > crude venom of the snake. > Also, the protein is expressed under alpha factor signal > sequence which is supposed to be secreted into the medium. > Therefore,once the protein is out into medium, it is out of > cell now, Kex2 and Ste13 cleavages should be happening > outside the cell, therefore, there should be no proteasomal > degradation. Am I right? > > After clipping off signal sequence, protein will be exported > out, still will there be any proteasomal targetting of > proteins with destabilizing residue at -N terminus? > > I read in -N end rule that, protein's life depends upon > penultimate residue to Met. We know, for a protein to be > synthesised, we need to have a start codon,i.e. Met. In -N > end rule they say, Met will be removed be > Metaminopeptidase(MAP), so if we express a protein we should > not be counting Met residue in the expected MW? Because, not > all mature protein sequences start from Met. > > > Then,I tried expressing my protein using E. coli epression > system, cDNA cloned in pET19b in Rosetta gami cells and RIL > cells.I couldn't see any expression. Cells were growing > normaly.(I hope, the protein is not inhibiting transcription > and translation). Met and Gly had to be added at -N terminus > to maintain the reading frame. > Protein is 10Kda and was cloned without tag sequence. I mean > expected protein should be 10kDa.I confirmed the sequences > before cloning and even plasmid isolated from ex-pression > hosts. DNA is there but no protein. > > Please answer any, if not all, of the above question. i am > struggling with all this for alst 8 months. Your suggestions > and help will be highly appreciable. > > Thanks in advance. > > Shifali > > > > Shifali Chatrath > Graduate Student > Protein science Lab > Dept. of Biological sciences > National University of Singapore > Singapore > +65-65161210 > +65-96393449 > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From editor from gene-quantification.info Wed Apr 29 09:18:03 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Thu Apr 30 14:40:45 2009 Subject: qPCR NEWS April 2009 - focus on microRNA normalisation Message-ID: <2baeeb47-67e7-4e8e-a492-9674898cc5f1@c18g2000prh.googlegroups.com> qPCR NEWS April 2009 - focus on microRNA normalisation -------------------------------------------------------------------------- Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - update in new microRNA reviews - NEW section about normalisation in microRNA experiments => http://www.gene-quantification.de/micro-rna-5.html#microrna-norm - Online translation service of the Gene Quantification - New qPCR events in autumn 2009 - New qPCR workshop modules at the TATAA Biocenter Germany - Online translation service of the Gene Quantification => http://translation.gene-quantification.info/ New qPCR workshop modules at the TATAA Biocenter Germany => http://tataa.gene-quantification.info/ European wide qPCR application workshops => register now ! => course program spring - summer 2009 => http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf -------------------------------------------------------------------------------- Data normalisation in microRNA experiments using qRT-PCR is a new challenge in gene quantification analysis. The reliability of any relative RT-PCR experiment can be improved by including an invariant endogenous control (reference gene) in the assay to correct for sample to sample variations in the qRT-PCR efficiency and errors in sample quantification. A biologically meaningful reporting of target mRNA copy numbers requires accurate and relevant normalisation to some standard and is strongly recommended in microRNA qRT-PCR. => But the quality of normalized quantitative expression data cannot be better than the quality of the normalizer itself. => Which are the best endogen microRNA normalizers? => Can we apply the same "microRNA normalizing strategy"? Any variation in the normalizer will obscure real changes and produce artifactual changes. Real-time RT-PCR-specific errors in the quantification of microRNA transcripts are easily compounded with any variation in the amount of starting material between the samples, e.g. caused by sample-to-sample variation and cDNA sample loading variation. This is especially relevant when the samples have been obtained from different individuals, different tissues and different time courses, and will result in the misinterpretation of the derived expression profile of the target genes. => Therefore, normalisation of target gene expression levels must be performed to compensate intra- and inter-kinetic RT-PCR variations (sample-to-sample & run-to-run variations). Data normalisation can be carried out against an endogenous unregulated reference gene transcript or against total cellular DNA or RNA content (molecules/g total DNA/RNA and concentrations/g total DNA/ RNA). Normalisation according the total cellular RNA content is increasingly used, but little is known about the total RNA content of cells or even about the microRNA or mRNA concentrations. The content per cell or per gram tissue may vary in different tissues in vivo, in cell culture (in vitro), between individuals and under different experimental conditions. Nevertheless, it has been shown that normalisation to total cellular RNA is the least unreliable method. It requires an accurate quantification of the isolated total RNA or mRNA or microRNA fraction by optical density at 260 nm, Lab-on-Chip capillary electrophoresis instruments, or Ribogreen RNA Quantification Kit. To normalize the absolute amount according to a single reference gene (or better a set of multiple stable reference genes), further sets of kinetic PCR reactions has to be performed for the invariant endogenous control(s) on all experimental samples and the relative abundance values are calculated for internal control as well as for the target gene. For each target gene sample, the relative abundance value obtained is divided by the value derived from the control sequence in the corresponding target gene. The normalized values for different biological samples can then directly be compared. The workflow: - check for good RNA integrity (=> http://RNA-integrity.gene-quantification.info/) - select stable internal reference microRNA or suitable smallRNAs (via Genorm or Normfinder) - calculate reference-gene-index of selected normalizers (geometric mean of Cq) - apply relative quantification strategy (comparable to mRNA relative quantification) - apply PCR efficiency correction (if wanted) - for microRNA normalistion strategies see papers below - or find some more ideas here => http://relative.gene-quantification.info => http://www.gene-quantification.de/micro-rna-5.html#microrna-norm -------------------------------------------------------------------------------- - Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues. - Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues. - Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. - High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. - Facile means for quantifying microRNA expression by real-time PCR. - A single-molecule method for the quantitation of microRNA gene expression. - Endogenous Controls for Real-Time Quantitation of miRNA Using TaqMan? MicroRNA Assays. -------------------------------------------------------------------------------- online translation Since October 2008 we provide an online translation service of the Gene Quantification pages to several languages. Please recognize this is an automatic and robotic based translation service, and therefore we can give NO guarantee about the generated content. It should help to understand the "rough" content of the Gene Quantification pages, but still the original is the ENGLISH version: http://translation.gene-quantification.info/ -------------------------------------------------------------------------------- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held regularly in G?teborg, Sweden, in English and in Freising-Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany workshops are held in cooperation with BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page: => http://TATAA.gene-quantification.info/ Course Occasions 2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) 3-day single-cell qPCR Module (Mon. - Wed.) 3-day microRNA Module (Mon. - Wed.) 20 - 22 April 2009 (E) NEW microRNA qPCR 11 - 15 May 2009 (Deutsch) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) 15 - 19 Juni 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) 13 - 15 Juli 2009 (E) NEW microRNA qPCR 27 - 31 Juli 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) => http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pdf -------------------------------------------------------------------------------- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. 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Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com The qPCR newsletter was end to [alle-adressen] To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=SUBSCRIBE From nik_duke1 from hotmail.com Thu Apr 30 17:43:44 2009 From: nik_duke1 from hotmail.com (Nik_ Duke1) Date: Thu Apr 30 18:11:12 2009 Subject: EDTA for lysozyme action In-Reply-To: <2021705c0904251151j13b0dd5axc1a946da0f405760@mail.gmail.com> References: <2021705c0904251151j13b0dd5axc1a946da0f405760@mail.gmail.com> Message-ID: Chiranjit, It would be most prudent to add EDTA and EGTA to protect proteolytic degradation of your proteins of interest, if you are trying to overexpress in E. coli, a Gram-negative bacteria. Release of highly active Aspartate proteases can chew up proteins very fast. Lysozyme activity is mildly reduced in the presence of chelator, but because it is a small molecule it can do away with upto 1mM EDTA. I know lysozyme is acting only on gram positive bacteria but the practice has been carried on Gram negative bacteria also. You may want to omit lysozyme step altogether if working with E.coli. Regards, Nik > Subject: EDTA for lysozyme action > > Can any one tell why EDTA is required for lysozyme action specially in case > of G- bacteria? What is the mechanism behind it? > > Regards > > -- > Chiranjit Chowdhury > Department of Biotechnology > Indian Institute of Technology, Kharagpur > Kharagpur, West Bengal, India > Pin: 721302 > Official email: chiranjit.chowdhury@iitkgp.ac.in > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods _________________________________________________________________ Rediscover Hotmail?: Get e-mail storage that grows with you. http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Storage2_042009