From Paul.Phelan from tufts.edu Thu Jul 2 15:05:51 2009 From: Paul.Phelan from tufts.edu (Paul J. Phelan) Date: Thu Jul 2 16:02:52 2009 Subject: Cracking 16% acrylamide SDS-PAGE gels Message-ID: <20090702160551.r0dofju92cwosgkw@webmail.tufts.edu> I think this is a trivial problem, but I have been trying to detect and resolve a cyclic peptide of mol. wt. 1,300 from a bacterial extract. I am using a peptide gel recipe for SDS-PAGE based on the Schagger + von Jagow Tris/Tricine system, that uses a 16% acrylamide resolving gel, a 9.5% acrylamide spacer gel, and a 5% acrylamide stacking gel. I'm having trouble detecting the peptide (it contains a 35S-Met), but my question is related to a problem we are having with the gels cracking after they are dried on a gel drier and then frozen at -80 C before being developed on x-ray film. Most of the time, the gels are cracked after drying, and freeze/thawing just makes the cracking worse. I think the gels are cracking just because they are more brittle at a high acrylamide concentration, but does anyone have any fool-proof tricks for drying thin (0.75 mm) 16% acrylamide mini-gels without cracking them? Any words of wisdom much appreciated, Paul Phelan From nick.theodorakis from gmail.com Fri Jul 3 16:19:57 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri Jul 3 18:09:04 2009 Subject: Cracking 16% acrylamide SDS-PAGE gels References: Message-ID: <542565ee-ce63-41c8-85fc-5116f39e19a9@n11g2000yqb.googlegroups.com> On Jul 2, 3:05=A0pm, "Paul J. Phelan" wrote: > I think this is a trivial problem, but I have been trying to detect and > resolve a cyclic peptide of mol. wt. 1,300 from a bacterial extract. =A0I > am using a peptide gel recipe for SDS-PAGE based on the Schagger + von > Jagow Tris/Tricine system, that uses a 16% acrylamide resolving gel, a > 9.5% acrylamide spacer gel, and a 5% acrylamide stacking gel. =A0I'm > having trouble detecting the peptide (it contains a 35S-Met), but my > question is related to a problem we are having with the gels cracking > after they are dried on a gel drier and then frozen at -80 C before > being developed on x-ray film. =A0Most of the time, the gels are cracked > after drying, and freeze/thawing just makes the cracking worse. > I think the gels are cracking just because they are more brittle at a > high acrylamide concentration, but does anyone have any fool-proof > tricks for drying thin (0.75 mm) 16% acrylamide mini-gels without > cracking them? > > Any words of wisdom much appreciated, > > Paul Phelan Unless you're doing fluorographic detection, you don't need to freeze the gel during exposure. I use a little=3Dknown gel recipe from Blattler et al (1972) that uses a variable acrylamide:bis ratio that seems to give gels with much better physical properties, such as resistance to cracking. The reference is: Blattler DP, Garner F, Van Slyke K, Bradley A. Quantitative electrophoresis in polyacrylamide gels of 2=9640%. J Chromatogr. 1972;64:147=96155. I also have a spreadsheet with formulas I can send you if you want to try it. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From ipek4477 from gmail.com Sat Jul 4 13:53:18 2009 From: ipek4477 from gmail.com (Ipek Bosgelmez) Date: Sat Jul 4 14:35:27 2009 Subject: Cracking 16% acrylamide SDS-PAGE gels Message-ID: <2896b49e0907041153y555be020q9ef64949e5e717f2@mail.gmail.com> I would be grateful if you could share these formulas against cracking gels. I usually work with 12.5% polyacrylamide gels, I hardly have a problem; but it would be nice to try some other recipe. Thanks in advance, Ipek On Sat, Jul 4, 2009 at 8:03 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. Re: Cracking 16% acrylamide SDS-PAGE gels (Nick Theodorakis) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Fri, 3 Jul 2009 14:19:57 -0700 (PDT) > From: Nick Theodorakis > Subject: Re: Cracking 16% acrylamide SDS-PAGE gels > To: methods@net.bio.net > Message-ID: > <542565ee-ce63-41c8-85fc-5116f39e19a9@n11g2000yqb.googlegroups.com> > Content-Type: text/plain; charset=windows-1252 > > On Jul 2, 3:05 pm, "Paul J. Phelan" wrote: > > I think this is a trivial problem, but I have been trying to detect and > > resolve a cyclic peptide of mol. wt. 1,300 from a bacterial extract. I > > am using a peptide gel recipe for SDS-PAGE based on the Schagger + von > > Jagow Tris/Tricine system, that uses a 16% acrylamide resolving gel, a > > 9.5% acrylamide spacer gel, and a 5% acrylamide stacking gel. I'm > > having trouble detecting the peptide (it contains a 35S-Met), but my > > question is related to a problem we are having with the gels cracking > > after they are dried on a gel drier and then frozen at -80 C before > > being developed on x-ray film. Most of the time, the gels are cracked > > after drying, and freeze/thawing just makes the cracking worse. > > I think the gels are cracking just because they are more brittle at a > > high acrylamide concentration, but does anyone have any fool-proof > > tricks for drying thin (0.75 mm) 16% acrylamide mini-gels without > > cracking them? > > > > Any words of wisdom much appreciated, > > > > Paul Phelan > > Unless you're doing fluorographic detection, you don't need to freeze > the gel during exposure. > > I use a little=known gel recipe from Blattler et al (1972) that uses a > variable acrylamide:bis ratio that seems to give gels with much better > physical properties, such as resistance to cracking. The reference is: > > Blattler DP, Garner F, Van Slyke K, Bradley A. Quantitative > electrophoresis in polyacrylamide gels of 2-40%. J Chromatogr. > 1972;64:147-155. > > I also have a spreadsheet with formulas I can send you if you want to > try it. > > Nick > > -- > Nick Theodorakis > nick_theodorakis@hotmail.com > contact form: > http://theodorakis.net/contact.html > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 50, Issue 2 > ************************************** > -- ?yi ?al??malar ve kolayl?klar dilerim. ?.?pek Bo?gelmez Ankara ?niversitesi Eczac?l?k Fak?ltesi F. Toksikoloji AD 06100, Tando?an-Ankara Tel: 0 312 203 31 21 Fax: 0 312 213 10 81 From vcook from wfubmc.edu Mon Jul 6 13:06:56 2009 From: vcook from wfubmc.edu (Vicky Cook) Date: Mon Jul 6 13:48:51 2009 Subject: cracking gels Message-ID: <9AEEF1FB6254224AA355ED285F84916538ACAD2A@EXCHVS2.medctr.ad.wfubmc.edu> I am not sure I can call this a fool-proof method, but back in the > days before imaging stations, when we routinely dried our acrylamide > gels, we used to us the following technique. After destaining in 10% > acetic acid, we soaked the gel in 2% glycerol. This can be done overnight. > Adding 0.02-0.05% sodium azide can preserve if you want to store the > gel a bit longer in the glycerol. Then we dried, and always tried to > use a gradual heating of the gel. Opt for longer drying with less > heat; this tends to prevent cracking. From peter.ianakiev from gmail.com Wed Jul 8 08:21:19 2009 From: peter.ianakiev from gmail.com (peter) Date: Wed Jul 8 12:38:27 2009 Subject: strand displacement activity assay Message-ID: <714ed4af-7f75-4c0b-be18-c1c50dcdd963@n11g2000yqb.googlegroups.com> Hello fellow scientists, I am trying to come up with an assay to determine strand displacement activity of T4 DNA pol. Any ideas? In general it is accepted that T4 pol has very little strand displacement, but for my application I need complete lack of such (single molecule assay). Thanks in advance old timers.... From aawara from pontiff-playground.org Wed Jul 8 09:33:59 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Wed Jul 8 12:38:32 2009 Subject: strand displacement activity assay References: <714ed4af-7f75-4c0b-be18-c1c50dcdd963@n11g2000yqb.googlegroups.com> Message-ID: In <714ed4af-7f75-4c0b-be18-c1c50dcdd963@n11g2000yqb.googlegroups.com>, peter wrote: > Hello fellow scientists, > I am trying to come up with an assay to determine strand displacement > activity of T4 DNA pol. Any ideas? In general it is accepted that T4 > pol has very little strand displacement, but for my application I need > complete lack of such (single molecule assay). Thanks in advance old > timers.... I've done such assays; I don't know if the sensitivity is suitable for your needs, but this is one way to get it done: a) Make a ssDNA closed circle which will be used as template. It is important that this be a closed circle, so that the 3'->5' exo of T4 doesn't chew it up. These days, I imagine you can make a long single-stranded oligo that has a modified 3'end, so that exonucleolytic degradation cannot occur. Use this long oligo as template. b) Anneal two oligos to it - one of which is radioactively labeled, and ends in a 3' phosphate, not 3' hydroxyl. This oligo must be about approximately 10 nucleotides 3' to then 3' end of the other oligo. c) Add T4 DNA pol and dNTPs. Electrophorese the reaction by non-denaturing PAGE of suitable percentage. d) The annealed radiolabeled oligo will run near the top of such a gel. Any displaced radiolabeled oligo will migrate faster. Good luck, A -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From peter.ianakiev from gmail.com Wed Jul 8 11:58:45 2009 From: peter.ianakiev from gmail.com (peter) Date: Wed Jul 8 14:00:26 2009 Subject: strand displacement activity assay References: <714ed4af-7f75-4c0b-be18-c1c50dcdd963@n11g2000yqb.googlegroups.com> Message-ID: On Jul 8, 10:33?am, Aawara Chowdhury wrote: > In <714ed4af-7f75-4c0b-be18-c1c50dcdd...@n11g2000yqb.googlegroups.com>, > > ?peter wrote: > > Hello fellow scientists, > > I am trying to come up with an assay to determine strand displacement > > activity of T4 DNA pol. Any ideas? In general it is accepted that T4 > > pol has very little strand displacement, but for my application I need > > complete lack of such (single molecule assay). Thanks in advance old > > timers.... > > I've done such assays; I don't know if the sensitivity is suitable for > your needs, but this is one way to get it done: > > a) Make a ssDNA closed circle which will be used as template. ?It is > ? ?important that this be a closed circle, so that the 3'->5' exo > ? ?of T4 doesn't chew it up. ?These days, I imagine you can make a > ? ?long single-stranded oligo that has a modified 3'end, so that > ? ?exonucleolytic degradation cannot occur. Use this long oligo > ? ?as template. > > b) Anneal two oligos to it - one of which is radioactively labeled, > ? ?and ends in a 3' phosphate, not 3' hydroxyl. > ? ?This oligo must be about approximately 10 nucleotides 3' to then > ? ?3' end of the other oligo. > > c) Add T4 DNA pol and dNTPs. ?Electrophorese the reaction by > ? ?non-denaturing PAGE of suitable percentage. > > d) The annealed radiolabeled oligo will run near the top of such > ? ?a gel. ?Any displaced radiolabeled oligo will migrate faster. > > Good luck, > A > -- > Email: echo 36434455860060025978157675027927670979097959886449930P | dc Thanks for the suggestion, Unfortunately I don't have permit for radioactivity in this lab....I was thinking something along annealing oligo that have a point mutation that would abolish restriction site and then making dsDNA with another oligo . If it gets displaced it will be cut with the RE, if it does not get cut - oligo was not displaced. That is assuming 100% annealing though .... From SBrown from ccia.unsw.edu.au Wed Jul 8 16:18:17 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Wed Jul 8 16:52:49 2009 Subject: luciferase reporter assay Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A07D3B6DD@mail01.ccia.local> Hi, Can anyone recommend a protocol for harvesting 293T to be usewd in a luciferase reporter assay. i have no experience with this tecnique, i have one construct with CMV in it and i expect this to be constituatively expressed, i have used this to guage how green the cells are going to be and therefore i have plated serial dilutions of cell # to work out the optimal cell density. I have a promotorless construct as a negative control etc etc What i am after is any tricks of the trade or advice as to how i go about taking these cells, specifically this step and then any information on how i go about setting up the cells in a white 96well plate to be analysed. Also can anyone recommend a supplier for the reagents, is promega the standard? Thanks in advance 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. 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 SBrown from ccia.unsw.edu.au Wed Jul 8 16:24:22 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Wed Jul 8 16:52:55 2009 Subject: Sybe Safe as a cell marker Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A07D3B6DE@mail01.ccia.local> Hi all, This may seem a little random, i know PI can be used with flow, i was wondering if sybr safe or ethidium bromide can be utilised in the same way. The biology is the same, both etbr and sybr safe intercollate withing the DNA, so any advice or theoretical ideas would be much appreciated, or is this idea plagued with errors? S 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. 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 aawara from pontiff-playground.org Wed Jul 8 23:29:40 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Thu Jul 9 08:35:03 2009 Subject: strand displacement activity assay References: <714ed4af-7f75-4c0b-be18-c1c50dcdd963@n11g2000yqb.googlegroups.com> Message-ID: In , peter wrote: > Thanks for the suggestion, > Unfortunately I don't have permit for radioactivity in this lab....I > was thinking something along annealing oligo that have a point > mutation that would abolish restriction site and then making dsDNA > with another oligo . If it gets displaced it will be cut with the RE, > if it does not get cut - oligo was not displaced. That is assuming > 100% annealing though .... If you have access to a UV-light box, you can FITC label the 3' end. This works, although the sensitivity is lower than 32P. It is more suitable for high-thruput applications. A -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From engelbert_buxbaum from hotmail.com Thu Jul 9 12:14:29 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Jul 9 14:06:53 2009 Subject: Cracking 16% acrylamide SDS-PAGE gels References: Message-ID: Am 02.07.2009, 17:45 Uhr, schrieb DK : >> a problem we are having with the gels cracking >> after they are dried on a gel drier and then frozen at -80 C before >> being developed on x-ray film. Most of the time, the gels are cracked >> after drying, and freeze/thawing just makes the cracking worse. >> I think the gels are cracking just because they are more brittle at a >> high acrylamide concentration, but does anyone have any fool-proof >> tricks for drying thin (0.75 mm) 16% acrylamide mini-gels without >> cracking them? >> >> Any words of wisdom much appreciated, > > Equilibrate the gels with 3% glycerol before drying. Then they > won't crack. One thing you can try in addition to using glycerol is to dry gels at room temperature. You place them between two sheets of wet cellulose acetate foil, those you span in a drying frame (BioRad floggs them, but they are just two rectangular frames of plastic, about 20 mm wide and 4 or 5 mm thick, held together by clamps). Those you put upright in a quiet place over night. As the sheets dry they shrink, and you get a flat, translucent dried gel that is also easy to rehydrate, if need be. Obviously, for weak radiators like 35-S you need to use autofluorography rather than autoradiography. From rahimpour_a from yahoo.com Tue Jul 14 00:57:02 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Tue Jul 14 11:38:45 2009 Subject: invitrogen flp-in Message-ID: <876568.43623.qm@web52705.mail.re2.yahoo.com> Dear All does any body use invitrogen flp in for establishment of stable cell lines? how is its efficiency? ? regards From rahimpour_a from yahoo.com Tue Jul 14 06:19:24 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Tue Jul 14 11:38:52 2009 Subject: flurescent proteins Message-ID: <530941.10524.qm@web52710.mail.re2.yahoo.com> hi I have a question about measuring fluorescence intensity of gfp, is it important to use the exact excitation emission wavelenght (488/509) or applying wavelenghts neat to that might works? does anyone can help? ? regards azam ? From Lionel.Fontao from hcuge.ch Tue Jul 14 06:21:14 2009 From: Lionel.Fontao from hcuge.ch (Lionel FONTAO) Date: Tue Jul 14 11:38:56 2009 Subject: RT-PCR problems on skin total RNA&In-Reply-To= Message-ID: <4A5C864B.DD74.0051.0@diogenes.hcuge.ch> I also had similar problems and was wondering whether you have any option stoolve this problem. Thanks for your help Lionel Fontao PhD From fulvio.celsi from gmail.com Tue Jul 14 11:55:37 2009 From: fulvio.celsi from gmail.com (Fulvio Celsi) Date: Tue Jul 14 14:16:48 2009 Subject: flurescent proteins In-Reply-To: <530941.10524.qm@web52710.mail.re2.yahoo.com> References: <530941.10524.qm@web52710.mail.re2.yahoo.com> Message-ID: Hi usually depends on your exc/abs filter...and also what kind of gfp are talking about...as a rule of thumb, I would say that =/- 15-20 nm is ok..for semi-quantitave measuring...for just visualizing gfp, you can go down to 450 or also 420 (exc) and 525-550 (em) In any case, use invitrogen spectra viewer http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluorescence-SpectraViewer.html to check the compatibility with your filtersets... best.. Fulvio Fulvio Celsi BsC,PhD Sector of Neurobiology, International School for Advanced Studies, Scuola Internazionale di Studi Superiori Avanzati (SISSA), Trieste Italy 2009/7/14 Azam Rahimpour > hi > I have a question about measuring fluorescence intensity of gfp, is it > important to use the exact excitation emission wavelenght (488/509) or > applying wavelenghts neat to that might works? does anyone can help? > > regards > azam > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From guanyuanfu from gmail.com Wed Jul 15 20:11:57 2009 From: guanyuanfu from gmail.com (guanyuan fu) Date: Wed Jul 15 21:10:10 2009 Subject: cleaning coverslip for protein/cell attachment Message-ID: Hi, all: I have some trouble with cleansing my coverslips for protein coating. The coverslips (22*22 mm) seemed to be very hydrophobic, as the protein solution (500 microliter) tended to aggregate into a big drop instead of disperse all over the surface of the coverslip. Normally, about 100 microliter solution should be able to cover the whole surface. I don't have a way to measure how much the protein is attached, but it seems to be very few if any based on the fact that my cells expressing activated receptor for that protein could not attach to the surface. The procedure is listed below: 1) Wash the coverslips with chromic sulfuric acid (fisherbrand cleaning solution) for overnight. 2) Rinse with running tape water for 3 hrs to remove chemical residues from step 1. 3) Rinse with distilled water 3 times each 30 mins. 4) Wash coverslips with 100% alcohol (mixed alcohol, not pure ethanol) for 2 hrs 5) Discard alcohol, and separate coverslips one by one to let it air dry. 6) After dry, store in glass plate then autoclave. Please give me any suggestion on modifying the above procedures since i am new to this methods. or if you have a good substitute (precleaned coverslip for protein coating to study cell spreading on the coated protein only), please also tell me where i can buy it. Thanks, Guanyuan Fu From novalidaddress from nurfuerspam.de Thu Jul 16 04:54:39 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Jul 16 07:24:42 2009 Subject: Biotin contamination of BSA Message-ID: Dear Experts, I noticed that "ordinary" BSA is unsuitable for blocking westerns when streptavidin HRP is used, as it causes high background by its biotin content. Where does the Biotin contamination come from and how may I get rid of it (except buying biotin free BSA)? Is it covalently linked? Best regards, Wo From ruchijainjnu from gmail.com Thu Jul 16 08:01:43 2009 From: ruchijainjnu from gmail.com (ruchi jain) Date: Thu Jul 16 11:53:33 2009 Subject: Biotin contamination of BSA In-Reply-To: References: Message-ID: <750ffe50907160601s73060cf4gf2ed1652ddae8f71@mail.gmail.com> Do the blocking with either gelatin or skimmed milk. We have been using skimmed milk and I would say its a cheap and a very good way of blocking in westerns. Best Ruchi On Thu, Jul 16, 2009 at 3:24 PM, WS wrote: > Dear Experts, > > I noticed that "ordinary" BSA is unsuitable for blocking westerns when > streptavidin HRP is used, as it causes high background by its biotin > content. Where does the Biotin contamination come from and how may I > get rid of it (except buying biotin free BSA)? Is it covalently > linked? > > Best regards, > > Wo > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Ruchi Jain, Ph.D. Postdoc Associate Internal Medicine-Infectious Diseases Yale University ruchi.jain@yale.edu 203-737-1197 From engelbert_buxbaum from hotmail.com Thu Jul 16 10:10:09 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Jul 16 11:53:39 2009 Subject: flurescent proteins References: Message-ID: Am 14.07.2009, 07:19 Uhr, schrieb Azam Rahimpour : > hi > I have a question about measuring fluorescence intensity of gfp, is it > important to use the exact excitation emission wavelenght (488/509) or > applying wavelenghts neat to that might works? does anyone can help? Peaks in optical spectroscopy are usually quite wide, so in principle you can use wavelengths "near" the maximum too. Obviously, the further you go away from the maximum, the lower the fluorescence observed. For a review on GFP see @article{Tsi-98, AUTHOR= {Tsien, Roger Y.}, TITLE= {The green fluorescent protein}, JOURNAL= {Annual Review of Biochemistry}, VOLUME= {67}, PAGES= {509-544}, YEAR= {1998}, DOI= {10.1146/annurev.biochem.67.1.509} } From vcook from wfubmc.edu Thu Jul 16 07:29:54 2009 From: vcook from wfubmc.edu (Vicky Cook) Date: Thu Jul 16 11:53:44 2009 Subject: (no subject) Message-ID: <9AEEF1FB6254224AA355ED285F84916538FBAA47@EXCHVS2.medctr.ad.wfubmc.edu> Hello, I am trying to produce TEV protease, and I have a few questions. First, why is DTT added to this enzyme? I think it has to do with its being a cysteine protease. Second, I am observing strands or strings in my dialysis bag, when I dialyze eluate from His bind column (in 20mM Tris-HCl pH 7.9, 500mM NaCl, 1M imidazole) into STB (10mM Tris-HCl pH 8.0, 150mM NaCl, 1mM EDTA, 0.02% sod. azide, 5mM DTT). I think it may have to do with either the salt concentration or the DTT? BTW, is it imidizole or imidazole? I have seen it written both ways. I think they are two different chemicals, but I am trained as a biologist, not too good with chemistry.... :) Thanks to all. From ruchijainjnu from gmail.com Thu Jul 16 08:06:31 2009 From: ruchijainjnu from gmail.com (ruchi jain) Date: Thu Jul 16 11:53:49 2009 Subject: cleaning coverslip for protein/cell attachment In-Reply-To: References: Message-ID: <750ffe50907160606n5f742307j809ecd65ca742fd9@mail.gmail.com> Hi, Although the protocol looks quite standard and similar one works for me but i quote my coverslips with polylysine at the end. In any case u can buy precleaned coverslips from various companies. Just search for it!!! A quick search gave me this link http://www.sorvall.com/com/cda/landingpage/0,10255,2852,00.html hope it will b of use to you!!! Best Ruchi On Thu, Jul 16, 2009 at 6:41 AM, guanyuan fu wrote: > Hi, all: > > I have some trouble with cleansing my coverslips for protein coating. The > coverslips (22*22 mm) seemed to be very hydrophobic, as the protein > solution > (500 microliter) tended to aggregate into a big drop instead of disperse > all > over the surface of the coverslip. Normally, about 100 microliter solution > should be able to cover the whole surface. I don't have a way to measure > how > much the protein is attached, but it seems to be very few if any based on > the fact that my cells expressing activated receptor for that protein could > not attach to the surface. > > The procedure is listed below: > > 1) Wash the coverslips with chromic sulfuric acid (fisherbrand cleaning > solution) for overnight. > 2) Rinse with running tape water for 3 hrs to remove chemical residues from > step 1. > 3) Rinse with distilled water 3 times each 30 mins. > 4) Wash coverslips with 100% alcohol (mixed alcohol, not pure ethanol) for > 2 > hrs > 5) Discard alcohol, and separate coverslips one by one to let it air dry. > 6) After dry, store in glass plate then autoclave. > > Please give me any suggestion on modifying the above procedures since i am > new to this methods. or if you have a good substitute (precleaned coverslip > for protein coating to study cell spreading on the coated protein only), > please also tell me where i can buy it. Thanks, > > Guanyuan Fu > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Ruchi Jain, Ph.D. Postdoc Associate Internal Medicine-Infectious Diseases Yale University ruchi.jain@yale.edu 203-737-1197 From engelbert_buxbaum from hotmail.com Thu Jul 16 10:46:38 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Jul 16 11:53:54 2009 Subject: cleaning coverslip for protein/cell attachment References: Message-ID: Am 15.07.2009, 21:11 Uhr, schrieb guanyuan fu : > Hi, all: > > I have some trouble with cleansing my coverslips for protein coating. The > coverslips (22*22 mm) seemed to be very hydrophobic, > The procedure is listed below: > > 1) Wash the coverslips with chromic sulfuric acid (fisherbrand cleaning > solution) for overnight. > 2) Rinse with running tape water for 3 hrs to remove chemical residues > from > step 1. > 3) Rinse with distilled water 3 times each 30 mins. > 4) Wash coverslips with 100% alcohol (mixed alcohol, not pure ethanol) > for 2 > hrs > 5) Discard alcohol, and separate coverslips one by one to let it air dry. > 6) After dry, store in glass plate then autoclave. chromium sulfuric acid you can make yourself by dissolving 10% potassium dichromate in conc. sulfuric acid (with the obious precautions). It is bright orange, and turns green when completely used up. In between, it looks brownish. I use a small volume of the acid, and then discard it after one time use. Place slips into the acid one by one, so that all surfaces are in contact with the chemical. Once they have been rinsed with water, place them into a holder that keeps them a mm apart, again to ensure that all surfaces are accessible to the solutions. Obviously, once slips come out of the acid, you can touch them only with tweezers, so that no new greese gets onto them. From engelbert_buxbaum from hotmail.com Thu Jul 16 10:50:59 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Thu Jul 16 11:53:59 2009 Subject: Biotin contamination of BSA References: Message-ID: Am 16.07.2009, 05:54 Uhr, schrieb WS : > I noticed that "ordinary" BSA is unsuitable for blocking westerns when > streptavidin HRP is used, as it causes high background by its biotin > content. Where does the Biotin contamination come from and how may I > get rid of it (except buying biotin free BSA)? Is it covalently > linked? Try fish skin (teleost) gelatin instead, or polyvinylpyrollidone (PVP). If you blot on PVDF membranes, you do not need to block at all. Air-dry the membrane completely, then all solutions can interact only with surface-bound proteins, but not with the very hydrophobic membrane -> no background binding. From yvonne.couch from pharm.ox.ac.uk Thu Jul 16 11:55:32 2009 From: yvonne.couch from pharm.ox.ac.uk (Yvonne Couch) Date: Thu Jul 16 14:54:58 2009 Subject: DMSO Alternative? Message-ID: <009601ca0636$3b650fc0$b22f2f40$@couch@pharm.ox.ac.uk> Hi all, I want to do some experiments in vivo with a peptide that is relatively hydrophobic, it will only go into solution after dissolving in a solvent, allowing evaporation to occur and then dissolving in DMSO. Is there an alternative solution to DMSO or an alternative method I could use to get the peptide into a solution that would be safe to use? Thanks Yvonne From nick.theodorakis from gmail.com Thu Jul 16 16:01:44 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Thu Jul 16 17:39:23 2009 Subject: (no subject) References: Message-ID: On Jul 16, 8:29?am, "Vicky Cook" wrote: > Hello, > > I am trying to produce TEV protease, and I have a few questions. ?First, > why is DTT added to this enzyme? I don't know about this enzyme specifically, but in general for enzymes that contain free sulfhydryl groups often DTT or some other reductant is included in its storage or reaction buffer to endure that it doesn't get oxidized. >?I think it has to do with its being a > cysteine protease. ?Second, I am observing strands or strings in my > dialysis bag, when I dialyze eluate from His bind column (in 20mM > Tris-HCl pH 7.9, 500mM NaCl, 1M imidazole) into STB (10mM Tris-HCl pH > 8.0, 150mM NaCl, 1mM EDTA, 0.02% sod. azide, 5mM DTT). ?I think it may > have to do with either the salt concentration or the DTT? ? It's not uncommon for a protein to precipitate when the salt concentration is changed. >BTW, is it > imidizole or imidazole? ?I have seen it written both ways. ?I think they > are two different chemicals, but I am trained as a biologist, not too > good with chemistry.... ?:) > Imidazole. Azoles are a class of compounds with five member rings containing nitrogen: Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From engelbert_buxbaum from hotmail.com Fri Jul 17 14:15:06 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jul 17 16:43:00 2009 Subject: DMSO Alternative? References: Message-ID: Am 16.07.2009, 12:55 Uhr, schrieb Yvonne Couch : > I want to do some experiments in vivo with a peptide that is relatively > hydrophobic, it will only go into solution after dissolving in a solvent, > allowing evaporation to occur and then dissolving in DMSO. Is there an > alternative solution to DMSO or an alternative method I could use to get > the peptide into a solution that would be safe to use? Increasing or decreasing the pH may bring the peptide into aqueous solution by increasing the number of charged amino acids. If that doesn't work, you have to experiment. Methanol or acetonitrile would be obvious candidates. Why do you want to replace the DMSO in the first place? That reason may well limit your options with other solvents as well. From SBrown from ccia.unsw.edu.au Fri Jul 17 18:53:09 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Sat Jul 18 13:31:24 2009 Subject: leptomycin B Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A07D3B6FB@mail01.ccia.local> Hi Guys, Has anyone had any experience witt leptomycin B (a crm-1 channel blocker) specifically with 293 cells? 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. 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 vasu.nanobiotech91 from gmail.com Sun Jul 19 13:01:05 2009 From: vasu.nanobiotech91 from gmail.com (Shri) Date: Sun Jul 19 13:39:57 2009 Subject: Help in Primer designing for expression... Message-ID: <845ed6a2-5138-4a4e-a376-3c41039af926@p10g2000prm.googlegroups.com> Hi friends.. I am working with pET vector system. for expression of the enzme in E.coli BL21. I have to design primer with Nco1 site in forward primer. so can anybody will suggest me about the placing of ATG site of ORF...whether it should be in the primer's R.E site or else again i have to add in the primer aftre the R.E site. waiting for suggetion from scientific community From hroychow from nmsu.edu Sun Jul 19 15:47:39 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Sun Jul 19 19:31:32 2009 Subject: Help in Primer designing for expression... In-Reply-To: <845ed6a2-5138-4a4e-a376-3c41039af926@p10g2000prm.googlegroups.com> References: <845ed6a2-5138-4a4e-a376-3c41039af926@p10g2000prm.googlegroups.com> Message-ID: <3045.67.41.8.229.1248036459.squirrel@webmail.nmsu.edu> Since the ATG start codon is part of the NCO1 sequence, we use that RE site for in-frame ligation. So, if your amplicon is digested and religated at the NCO1 site, you should be fine. However, it has always been my advise that the primary amplified product be first cloned into a simple vector (pUC based) for obtaining ample inserts for a ligation. Following that, the insert may be digested out with the compatible RE (in your case, NCO1). > Hi friends.. > > I am working with pET vector system. for expression of the enzme in > E.coli BL21. > I have to design primer with Nco1 site in forward primer. > so can anybody will suggest me about the placing of ATG site of > ORF...whether it should be in the primer's R.E site or else again i > have to add in the primer aftre the R.E site. > > waiting for suggetion from scientific community > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From ricc_wparma from yahoo.it Sun Jul 19 15:47:44 2009 From: ricc_wparma from yahoo.it (riccardo senter) Date: Sun Jul 19 19:31:46 2009 Subject: protein extraction Message-ID: <237478.75652.qm@web23203.mail.ird.yahoo.com> hi! i'm a new entry. i need to extract proteins?from PBMC in order to perform a western blot and a functional assay of iNOS, which?is a cytosolic homodimeric protein. which is the best extraction?protocol? Another question: can i use PBMC pellets that?have been?storing?for several years at -20?? From adilabbasi7581 from yahoo.co.in Mon Jul 20 06:05:23 2009 From: adilabbasi7581 from yahoo.co.in (Adil A.Abbasi) Date: Mon Jul 20 09:06:53 2009 Subject: Kind Attent Message-ID: <357961.23594.qm@web95207.mail.in2.yahoo.com> All Dear Scientist Please help me out, I am a student (M.Sc.) I have to do an assay of Sodium Ascorbyl Phosphate. Can any One please tell me How I check Assay of ascorbic acid in it. Thanking you with Anticipation. Looking for local information? Find it on Yahoo! Local http://in.local.yahoo.com/ From shifalich from rediffmail.com Mon Jul 20 02:12:16 2009 From: shifalich from rediffmail.com (shifalichatrath) Date: Mon Jul 20 09:07:01 2009 Subject: Help in Primer designing for expression... Message-ID: <1248029009.S.3227.35605.f4mail-235-130.rediffmail.com.1248073936.26022@webmail.rediffmail.com> Dear Vasu! If you are adding NcoI site in FP, When after digestion of your insert amplified from what so ever template, you can clone it into vector digested with NcoI. So, actually, your Nco I site will remain intact after ligation and you will have ATG and this will be a part of your primer. But, before you order primer, you should make sure that starting from ATG your construct is in correct reading frame. As, Nco I site is CCATGG (I think, i didn't cross check.)Therefore, add two extra nucleotides GG or GC also to maintain your frame and you will have extra MG before start of your actual protein sequence. I hope this is what you asked! your question was not very clear. All the best. Shifali On Mon, 20 Jul 2009 00:13:29 +0530 wrote >Hi friends.. > >I am working with pET vector system. for expression of the enzme in >E.coli BL21. >I have to design primer with Nco1 site in forward primer. >so can anybody will suggest me about the placing of ATG site of >ORF...whether it should be in the primer's R.E site or else again i >have to add in the primer aftre the R.E site. > >waiting for suggetion from scientific community >_______________________________________________ >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 novalidaddress from nurfuerspam.de Mon Jul 20 02:18:42 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Mon Jul 20 09:07:07 2009 Subject: protein extraction References: Message-ID: On 19 Jul., 22:47, riccardo senter wrote: > hi! > i'm a new entry. Welcome! > i need to extract proteins?from PBMC in order to perform a western blot and a functional assay of iNOS, which?is a cytosolic homodimeric protein. > which is the best extraction?protocol? Basically, any buffer without SDS should do it. If in doubt, compare all protocols / buffers you may get hold of with a functional assay. My preferred start (without having checked any literature) would be 0.1% Triton X 100, Tween 20 or NP40 in PBS with protease inhibitors, cold, with a rotor-stator homogenizer. Detergent would be just for efficient lysis of cell walls, as iNOS is supposed to be cytosolic, and might be omitted if they should . For SDS-PAGE, mix aliquots with several times concentrated Laemmli style buffer and freeze. Check protein content with some other aliquot, then adjust the amount of protein in PAGE by sample volume. > > Another question: can i use PBMC pellets that?have been?storing?for several years at -20?? Difficult to say. I would doubt that any phosphorylation signal (if still present at all) is reliable. For protein integrity at all, compare with fresh samples. Good luck and best regards, Wo From blackhole from abuse.plus.com Mon Jul 20 08:22:01 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Mon Jul 20 09:07:14 2009 Subject: Help in Primer designing for expression... References: <845ed6a2-5138-4a4e-a376-3c41039af926@p10g2000prm.googlegroups.com> Message-ID: Historians believe that in newspost on Sun, 19 Jul 2009, Dr. Hiranya S. Roychowdhury penned the following literary masterpiece: >Since the ATG start codon is part of the NCO1 sequence, we use that RE >site for in-frame ligation. So, if your amplicon is digested and >religated at the NCO1 site, you should be fine. Just remember that for Nco I, the codon following the ATG must start with a G either original or you must add one i.e. CC - ATG - GNN Also pick the correct pET21 i.e. pET21d :-) For this reason I much prefer to use Nde I whenever possible i.e. CATATG. No problems with extra aa's. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From aawara from pontiff-playground.org Mon Jul 20 08:43:01 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Jul 20 09:07:20 2009 Subject: Help in Primer designing for expression... References: <845ed6a2-5138-4a4e-a376-3c41039af926@p10g2000prm.googlegroups.com> Message-ID: In , Duncan Clark wrote: > Historians believe that in newspost > on Sun, 19 Jul 2009, > Dr. Hiranya S. Roychowdhury penned the following > literary masterpiece: >>Since the ATG start codon is part of the NCO1 sequence, we use that RE >>site for in-frame ligation. So, if your amplicon is digested and >>religated at the NCO1 site, you should be fine. > > Just remember that for Nco I, the codon following the ATG must start > with a G either original or you must add one i.e. CC - ATG - GNN > > Also pick the correct pET21 i.e. pET21d :-) > > For this reason I much prefer to use Nde I whenever possible i.e. > CATATG. No problems with extra aa's. NcoI can be fairly versatile as well. An NcoI overhang is compatible with BspHI (TCATGA) and PciI (ACATGT). So, the second codon (after ATG) can begin with G (NcoI), A(BspHI), or T(PciI). Between these three, the second amino-acid can be anything other than histidine and proline. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From stewjw from gmail.com Mon Jul 20 05:25:13 2009 From: stewjw from gmail.com (StewJW) Date: Mon Jul 20 09:07:30 2009 Subject: DMSO Alternative? References: Message-ID: <478cd5b2-4605-4948-a97d-7d72c96ad81b@26g2000yqk.googlegroups.com> A few drops of acetonitrile is the obvious choice or DMF Changing thr pH as mentioned is another way. By the way, although extremely hydroscopic, smelling awful, and a bit of a sod to remove, DMSO is not toxic. From vcook from wfubmc.edu Mon Jul 20 10:29:07 2009 From: vcook from wfubmc.edu (Vicky Cook) Date: Mon Jul 20 13:09:25 2009 Subject: TEV Message-ID: <9AEEF1FB6254224AA355ED285F849165391772D9@EXCHVS2.medctr.ad.wfubmc.edu> Thanks, DK, for your good advice on rTEV. I suspected as much regarding the cysteine and imidAzole. Regarding the concentration of imidazole, I thought 1M was a lot too, but that is what the resin manufacturer and our plasmid donor have in their protocols. They use Novagen's His Bind resin for his tag removal. The elution buffer is 0.5M. Our donor uses bind (or lysis), bind and elution buffers with added 10% glycerol as well, but they do not add it to dialysis (?). I did, but it still precipitated. My PI wants me to try concentrating in elution buffer by stirred cell, and then dialyzing. I wanted to clarify the DTT: you mean use TCEP instead of DTT, or the other way around? Thanks, Vicky From arnec from bio.umass.edu Mon Jul 20 14:35:38 2009 From: arnec from bio.umass.edu (Arne K Christensen) Date: Mon Jul 20 22:48:45 2009 Subject: Homemade chemifluorescence Message-ID: <4A64C70A.9030403@bio.umass.edu> Dear listeners, Our lab is considering a switch from enhanced chemiluminescence (ECL) to chemifluorescence (ECF), as we may soon have access to a Storm 860 phosphorimager. The folks at Molecular Dynamics suggest ECF over ECL for HRP detection with the Storm 860. What is troubling is that our lab would have to abandon its cheap and effective homemade ECL protocol consisting of H2O2 + (luminol + coumaric acid). Does anybody know of a cheap and effective homemade ECF protocol? Thanks in advance, Arne Christensen -- __ Arne K Christensen Postdoctoral Research Associate University of Massachusetts, Amherst USGS Conte Anadromous Fish Research Center One Migratory Way, PO Box 796 Turners Falls, MA 01376 Email: arnec@bio.umass.edu Phone: (413) 863-3827 Fax: (413) 863-9810 URL: www.biobog.com From novalidaddress from nurfuerspam.de Tue Jul 21 03:43:15 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Tue Jul 21 08:39:27 2009 Subject: [a bit OT] floating "precipitates" Message-ID: Dear Experts, please excuse my ignorance: How to describe stuff that goes out of solution, but does not precipitate at the bottom of the vial and prefers floating atop of it - Floating insoluble matter? Flotulate? Wo From engelbert_buxbaum from hotmail.com Tue Jul 21 08:04:42 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jul 21 08:39:32 2009 Subject: DMSO Alternative? References: <478cd5b2-4605-4948-a97d-7d72c96ad81b@26g2000yqk.googlegroups.com> Message-ID: Am 20.07.2009, 06:25 Uhr, schrieb StewJW : > A few drops of acetonitrile is the obvious choice or DMF Changing thr > pH as mentioned is another way. By the way, although extremely > hydroscopic, smelling awful, and a bit of a sod to remove, DMSO is not > toxic. Sure. However, it is skin permeable and can take dissolved substances with it. So the habit of dissolving toxic substances like PMSF in DMSO should be frowned upon. From engelbert_buxbaum from hotmail.com Tue Jul 21 08:23:28 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jul 21 15:43:12 2009 Subject: protein extraction References: Message-ID: Am 19.07.2009, 16:47 Uhr, schrieb riccardo senter : > hi! > i'm a new entry. > i need to extract proteins?from PBMC in order to perform a western blot > and a functional assay of iNOS, which?is a cytosolic homodimeric protein. > which is the best extraction?protocol? > > Another question: can i use PBMC pellets that?have been?storing?for > several years at -20?? I would simply start with a hypotonic buffer (20 mM HEPES-KOH pH 7 or the like) with the usual protease inhibitor coctail and pass the cells through a fine (27G) needle a couple of times. Then spin down nuclei, membranes and the like in an Eppendorf centrifuge (refrigerated). 2 min on full speed should do it. The supernatant contains the soluble proteins. If that does not work, you need to add detergents to your buffer. A very effective way is to use CTAB, then use cationic electrophoresis and eastern blotting for detection (Anal. Biochem. 314 (2003) 70-6). CTAB is even more effective in solubilisation than SDS, yet at the same time so gentle that it is often possible to run zymograms. As to the old samples, if they have been prepared with protease inhibitors, snap-frozen, constantly stored at low temperatures (no self-defrosting fridges!) and if you want to detect only protein, not post-translational modification or activity, it should probably work, especially if you have samples of known target concentration stored in the same way as controls. From engelbert_buxbaum from hotmail.com Tue Jul 21 08:28:13 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Jul 21 15:43:21 2009 Subject: [a bit OT] floating "precipitates" References: Message-ID: Am 21.07.2009, 04:43 Uhr, schrieb WS : > please excuse my ignorance: How to describe stuff that goes out of > solution, but does not precipitate at the bottom of the vial and > prefers floating atop of it - Floating insoluble matter? Flotulate? "Precipitate" refers to the material that has become insoluble during a process. Whether the density of that material is higher or lower than that of the solution has nothing to do with it. If the material has a higher density than the solution, and is allowed to settle (possibly with the aid of a centrifuge) you get a "pellet". From fengzeng0827 from gmail.com Tue Jul 21 08:47:06 2009 From: fengzeng0827 from gmail.com (feng zeng) Date: Tue Jul 21 15:43:29 2009 Subject: What is the working concentration of N-succinyl-(Ala)3-Nitroanilide when doing Elastase assay Message-ID: <2a82b3da0907210647u7991e3f2jc626dc6062494dd@mail.gmail.com> Hi, Do any people know what is the work concentration of N-succinyl-(Ala)3-Nitroanilide when doing Elastase assay? And what will be the colour when this substrate reacts with Elastase? I use the concentration 4.4mM, and and 20ul to make finally 200ul mixture of Elastase, Buffer and Substrate, and then measure the OD at 410nm I did not see good result:( Thank you all Feng From pow.joshi from gmail.com Tue Jul 21 16:06:52 2009 From: pow.joshi from gmail.com (Pow Joshi) Date: Tue Jul 21 16:13:35 2009 Subject: [a bit OT] floating "precipitates" In-Reply-To: References: Message-ID: <710764ea0907211406g4f0b2ad1l722e2cb38336770c@mail.gmail.com> awesome.. that's the one .... so I checked the origin on OED, and they say the origin of the word is "flocculus" or a flock of wool....thanks Dima 2009/7/21 DK > In article < > bca801ba-22dd-48d9-9073-ae4fb9dafd18@a7g2000yqk.googlegroups.com>, WS < > novalidaddress@nurfuerspam.de> wrote: > >Dear Experts, > > > >please excuse my ignorance: How to describe stuff that goes out of > >solution, but does not precipitate at the bottom of the vial and > >prefers floating atop of it - Floating insoluble matter? Flotulate? > > I think it's flocculate. > > DK > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From agbiok4 from gmail.com Wed Jul 22 05:35:55 2009 From: agbiok4 from gmail.com (kamalaker nasani) Date: Wed Jul 22 10:16:42 2009 Subject: Cultures Message-ID: Hi, I have to use XL1 BLUE Just I want to know the difference in terms of transformation efficiency and protocol. What antibiotic we need to use the XL1 BLUE -- Kamalaker Nasani, Biotechnologist, Global Transgenes Ltd. From dofernand from yahoo.com.br Wed Jul 22 12:24:37 2009 From: dofernand from yahoo.com.br (Scientist) Date: Wed Jul 22 19:56:15 2009 Subject: can protein modifications affect immunodetection? Message-ID: <5b6700d8-7e4c-422d-8673-dbba14a7cbad@h11g2000yqb.googlegroups.com> Hi all! im trying to detect a protein in plants Crude extract using polyclonal Abs. These Abs were obtained from rat sorum, The protein used to innolculate the rats was expressed in E.coli (thus lacking the typical postrasductional modifications occurring in plants) . Untill only no positve results were obtained. When recombinant form is used as positive control everthing works well. So the possibilities are : a) native form is absent in the plant. b) the protien is present but in low concetration c) native form is modified in someway , thus the abs cannot recognize the epitopes. have anyone a suggestions for these items? thanks a lot From virashkgupta from gmail.com Wed Jul 22 22:57:16 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Thu Jul 23 01:35:19 2009 Subject: Methods Digest, Vol 50, Issue 15-Cultures Message-ID: For XL-1-Blue use Tetracycline 5 microgram/ml. On 7/22/09, methods-request@oat.bio.indiana.edu < methods-request@oat.bio.indiana.edu> 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. Re: [a bit OT] floating "precipitates" (DK) > 2. Re: protein extraction (Dr Engelbert Buxbaum) > 3. Re: [a bit OT] floating "precipitates" (Dr Engelbert Buxbaum) > 4. What is the working concentration of > N-succinyl-(Ala)3-Nitroanilide when doing Elastase assay (feng > zeng) > 5. Re: TEV (DK) > 6. Re: [a bit OT] floating "precipitates" (Pow Joshi) > 7. Cultures (kamalaker nasani) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Tue, 21 Jul 2009 13:20:22 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: [a bit OT] floating "precipitates" > To: methods@net.bio.net > Message-ID: > > In article < > bca801ba-22dd-48d9-9073-ae4fb9dafd18@a7g2000yqk.googlegroups.com>, WS < > novalidaddress@nurfuerspam.de> wrote: > >Dear Experts, > > > >please excuse my ignorance: How to describe stuff that goes out of > >solution, but does not precipitate at the bottom of the vial and > >prefers floating atop of it - Floating insoluble matter? Flotulate? > > I think it's flocculate. > > DK > > > ------------------------------ > > Message: 2 > Date: Tue, 21 Jul 2009 09:23:28 -0400 > From: "Dr Engelbert Buxbaum" > Subject: Re: protein extraction > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 19.07.2009, 16:47 Uhr, schrieb riccardo senter : > > > hi! > > i'm a new entry. > > i need to extract proteins from PBMC in order to perform a western blot > > and a functional assay of iNOS, which is a cytosolic homodimeric protein. > > which is the best extraction protocol? > > > > Another question: can i use PBMC pellets that have been storing for > > several years at -20?? > > I would simply start with a hypotonic buffer (20 mM HEPES-KOH pH 7 or the > like) with the usual protease inhibitor coctail and pass the cells through > a fine (27G) needle a couple of times. Then spin down nuclei, membranes > and the like in an Eppendorf centrifuge (refrigerated). 2 min on full > speed should do it. The supernatant contains the soluble proteins. > > If that does not work, you need to add detergents to your buffer. A very > effective way is to use CTAB, then use cationic electrophoresis and > eastern blotting for detection (Anal. Biochem. 314 (2003) 70-6). CTAB is > even more effective in solubilisation than SDS, yet at the same time so > gentle that it is often possible to run zymograms. > > As to the old samples, if they have been prepared with protease > inhibitors, snap-frozen, constantly stored at low temperatures (no > self-defrosting fridges!) and if you want to detect only protein, not > post-translational modification or activity, it should probably work, > especially if you have samples of known target concentration stored in the > same way as controls. > > > ------------------------------ > > Message: 3 > Date: Tue, 21 Jul 2009 09:28:13 -0400 > From: "Dr Engelbert Buxbaum" > Subject: Re: [a bit OT] floating "precipitates" > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; format=flowed; delsp=yes; > charset=iso-8859-15 > > Am 21.07.2009, 04:43 Uhr, schrieb WS : > > > please excuse my ignorance: How to describe stuff that goes out of > > solution, but does not precipitate at the bottom of the vial and > > prefers floating atop of it - Floating insoluble matter? Flotulate? > > "Precipitate" refers to the material that has become insoluble during a > process. Whether the density of that material is higher or lower than that > of the solution has nothing to do with it. > > If the material has a higher density than the solution, and is allowed to > settle (possibly with the aid of a centrifuge) you get a "pellet". > > > ------------------------------ > > Message: 4 > Date: Tue, 21 Jul 2009 09:47:06 -0400 > From: feng zeng > Subject: What is the working concentration of > N-succinyl-(Ala)3-Nitroanilide when doing Elastase assay > To: Methods@magpie.bio.indiana.edu > Message-ID: > <2a82b3da0907210647u7991e3f2jc626dc6062494dd@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, > > Do any people know what is the work concentration of > N-succinyl-(Ala)3-Nitroanilide when doing Elastase assay? > And what will be the colour when this substrate reacts with Elastase? > > I use the concentration 4.4mM, and and 20ul to make finally 200ul mixture > of > Elastase, Buffer and Substrate, and then measure the OD at 410nm > I did not see good result:( > > Thank you all > Feng > > > ------------------------------ > > Message: 5 > Date: Tue, 21 Jul 2009 15:23:58 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: TEV > To: methods@net.bio.net > Message-ID: > > In article , "Vicky > Cook" wrote: > >I wanted to clarify the DTT: you mean > >use TCEP instead of DTT, or the other way around? > > Yes, use TCEP instead of DTT. > > DK > > > ------------------------------ > > Message: 6 > Date: Tue, 21 Jul 2009 17:06:52 -0400 > From: Pow Joshi > Subject: Re: [a bit OT] floating "precipitates" > To: DK > Cc: methods@magpie.bio.indiana.edu > Message-ID: > <710764ea0907211406g4f0b2ad1l722e2cb38336770c@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > awesome.. that's the one .... so I checked the origin on OED, and they say > the origin of the word is "flocculus" or a flock of wool....thanks Dima > > 2009/7/21 DK > > > In article < > > bca801ba-22dd-48d9-9073-ae4fb9dafd18@a7g2000yqk.googlegroups.com>, WS < > > novalidaddress@nurfuerspam.de> wrote: > > >Dear Experts, > > > > > >please excuse my ignorance: How to describe stuff that goes out of > > >solution, but does not precipitate at the bottom of the vial and > > >prefers floating atop of it - Floating insoluble matter? Flotulate? > > > > I think it's flocculate. > > > > DK > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > ------------------------------ > > Message: 7 > Date: Wed, 22 Jul 2009 16:05:55 +0530 > From: kamalaker nasani > Subject: Cultures > To: methods@magpie.bio.indiana.edu, Plant Tissue Culture > , plantbio@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, > > I have to use XL1 BLUE > > Just I want to know the difference in terms of transformation efficiency > and > protocol. > > What antibiotic we need to use the XL1 BLUE > > -- > Kamalaker Nasani, > Biotechnologist, > Global Transgenes Ltd. > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 50, Issue 15 > *************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From bjornjobb from gmail.com Thu Jul 23 03:31:23 2009 From: bjornjobb from gmail.com (Bjorn) Date: Thu Jul 23 12:26:44 2009 Subject: E. coli mutator strain Message-ID: <5d4967c4-cd1c-40fc-a8c6-50934e7fa500@d32g2000yqh.googlegroups.com> Hi, I would like to try to do random mutagenesis with an E. coli mutator strain (such as XL1-red). I wonder if someone could send me some, i would rater not like to buy them since they are expensive and some people report too low mutation rates. Thanks, Bjorn From yvonne.couch from pharm.ox.ac.uk Thu Jul 23 03:57:57 2009 From: yvonne.couch from pharm.ox.ac.uk (Yvonne Couch) Date: Thu Jul 23 12:26:51 2009 Subject: Standard Curve qPCR Message-ID: <028a01ca0b73$ac934e40$05b9eac0$@couch@pharm.ox.ac.uk> Hi all, I have come against some contention as regards making a standard curve for qPCR. My current method is to take a known amount of cDNA (e.g. 40ng/ul) from each sample (so 2ul from 10 samples = total of 800ng) then dilute so I have 100ng total cDNA in my top standard, 50ng in the next, 25ng in the next, etc. However, a colleague PCRs up the exact region she needs (e.g. IL6) and then dilutes that as a standard for each set of primers. I was told the way I do things would be frowned upon if I tried to publish. Any thoughts? Cheers! From blackhole from abuse.plus.com Thu Jul 23 04:25:13 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Thu Jul 23 12:26:57 2009 Subject: Cultures References: Message-ID: Historians believe that in newspost on Wed, 22 Jul 2009, kamalaker nasani penned the following literary masterpiece: >What antibiotic we need to use the XL1 BLUE First find the genotype for XL1-BLUE. Google is your friend. Use that information plus a LOT of background reading to make YOUR decision. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From R.Jayakumar from roswellpark.org Thu Jul 23 08:39:43 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Thu Jul 23 12:27:08 2009 Subject: can protein modifications affect immunodetection? In-Reply-To: <5b6700d8-7e4c-422d-8673-dbba14a7cbad@h11g2000yqb.googlegroups.com> References: <5b6700d8-7e4c-422d-8673-dbba14a7cbad@h11g2000yqb.googlegroups.com> Message-ID: All three are strong possibilities. C is probably right. You should try developing monoclonal antibodies with recombinant protein and screening them for specificity against the native form. To check "b", try Immunoprecipitating the protein with the antibody. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Scientist Sent: Wednesday, July 22, 2009 1:25 PM To: methods@magpie.bio.indiana.edu Subject: can protein modifications affect immunodetection? Hi all! im trying to detect a protein in plants Crude extract using polyclonal Abs. These Abs were obtained from rat sorum, The protein used to innolculate the rats was expressed in E.coli (thus lacking the typical postrasductional modifications occurring in plants) . Untill only no positve results were obtained. When recombinant form is used as positive control everthing works well. So the possibilities are : a) native form is absent in the plant. b) the protien is present but in low concetration c) native form is modified in someway , thus the abs cannot recognize the epitopes. have anyone a suggestions for these items? thanks a lot _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From pattisyang from gmail.com Thu Jul 23 21:24:50 2009 From: pattisyang from gmail.com (Xuan Yang) Date: Thu Jul 23 21:34:36 2009 Subject: Confused about the different principles to freeze & thaw cells and purified protein Message-ID: Dear Sir or Madam, When we store cells (especially eukaryotic cells), storage box filled with isopropyl alcohol was used to prevent the temperature from dropping too fast. While thawing the cells from liquid nitrogen, we put the tube immediately into 37?C water bath. Freezing slowly, thawing fast. This strategy was totally contrary to the way we deal with purified proteins, namely freezing rapidly (favorablely in liquid nitrogen), but thawing slowly (favorablely on ice). It was quite confusing and I was wondering whether anyone would be so kind to offer me some explanations. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 We will either find a way or make one. From Zhonglin.Chai from bakeridi.edu.au Thu Jul 23 22:19:15 2009 From: Zhonglin.Chai from bakeridi.edu.au (Zhonglin Chai) Date: Thu Jul 23 22:31:18 2009 Subject: Confused about the different principles to freeze & thaw cells and purified protein In-Reply-To: References: Message-ID: <217613C88C00D4448B099AD7A374F2530BD34A60@exch01.bhri.internal> My personal view on this is that for cells, we tried to avoid formation of ice crystals which break cell membrane to kill your cells. This is achieved by the protocol you described with slow freezing (in DMSO) and fast thawing in 37oC degree water bath. For proteins, particularly for enzymes, or other bioactive samples, they may be sensitive to high temperatures, the best way to thaw is on ice to avoid exposing them to an undesirable higher temperature such as room temperature. Z Chai -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Xuan Yang Sent: Friday, 24 July 2009 12:25 PM To: methods@magpie.bio.indiana.edu Subject: Confused about the different principles to freeze & thaw cells and purified protein Dear Sir or Madam, When we store cells (especially eukaryotic cells), storage box filled with isopropyl alcohol was used to prevent the temperature from dropping too fast. While thawing the cells from liquid nitrogen, we put the tube immediately into 37?C water bath. Freezing slowly, thawing fast. This strategy was totally contrary to the way we deal with purified proteins, namely freezing rapidly (favorablely in liquid nitrogen), but thawing slowly (favorablely on ice). It was quite confusing and I was wondering whether anyone would be so kind to offer me some explanations. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 We will either find a way or make one. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. ______________________________________________________________________ This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you have received this email in error please promptly delete this email and notify the system manager. This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email From MConnelly from lab901.com Fri Jul 24 03:16:51 2009 From: MConnelly from lab901.com (Matthew Connelly) Date: Fri Jul 24 07:12:32 2009 Subject: Confused about the different principles to freeze & thaw cells and purified protein In-Reply-To: References: Message-ID: <5A6456E670F5564EB7095EC34563B39DE4AF51@lab901-svr01.Lab901.Com> When you freeze aqueous solutions slowly you form larger ice crystals. This would segregate water away from proteins and into the crystals of ice. As the amount of unfrozen water drops the protein becomes more and more concentrated and loses the water shell required to maintain its structure (remember the primary force for protein structure is the organisation of hydrophobic and hydrophilic residues). This quickly leads to protein denaturation and aggregation, and is one of the reasons why freeze thaw cycles are so damaging to proteins. Proteins are frozen quickly to produce the smallest ice crystals possible as quickly as possible, which minimises the disruption described above. Formulation of the protein with excipients such as polyols and carbohydrates can also help dramatically improve stability during freeze thaw by helping to lower the total amount of water bound up in ice crystals, and to take the place of water at the proteins surface. This can allow you to freeze proteins more slowly without causing damage. Not sure about cells, but I think that the freezing rates necessary for cryopreservation are actually dependent on the cell/tissue type... Matt -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Xuan Yang Sent: 24 July 2009 03:25 To: methods@magpie.bio.indiana.edu Subject: Confused about the different principles to freeze & thaw cells and purified protein Dear Sir or Madam, When we store cells (especially eukaryotic cells), storage box filled with isopropyl alcohol was used to prevent the temperature from dropping too fast. While thawing the cells from liquid nitrogen, we put the tube immediately into 37?C water bath. Freezing slowly, thawing fast. This strategy was totally contrary to the way we deal with purified proteins, namely freezing rapidly (favorablely in liquid nitrogen), but thawing slowly (favorablely on ice). It was quite confusing and I was wondering whether anyone would be so kind to offer me some explanations. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 We will either find a way or make one. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From pattisyang from gmail.com Fri Jul 24 04:35:20 2009 From: pattisyang from gmail.com (Xuan Yang) Date: Fri Jul 24 07:12:40 2009 Subject: Fwd: Confused about the different principles to freeze & thaw cells and purified protein In-Reply-To: References: <217613C88C00D4448B099AD7A374F2530BD34A60@exch01.bhri.internal> Message-ID: ---------- Forwarded message ---------- From: Xuan Yang Date: 2009/7/24 Subject: Re: Confused about the different principles to freeze & thaw cells and purified protein To: Zhonglin Chai Dear Mr or Ms Chai, Thanks for the reply and based on your explanation, the key difference if not the only difference should be that cells have fragile membrane while purified proteins do not. Slow freezing with DMSO (DMSO is critical and thanks for reminding me of that) should minimize the damage to cell membrane, while rapid freezing with glycerol should maximally retain the activity of purified proteins. Since I have no idea about what happened in the process of freezing and thawing, which seemed like an independent and mysterious filed itself, the exact reasons underlying these processes remained elusive to me. Then I did a little google about freezing and thawing. Based on a literature titled "Protein Denaturation during Freezing and Thawing in Phosphate Buffer Systems: Monomeric and Tetrameric ?-Galactosidase", at least pH change was involved in the process of freezing and thawing. Moreover, "thawing fast" seemed like a general principle applied to both cells and purified proteins. Thus if the protein sample was not heat sensitive, thawing on ice should be disfavored. Sincerely, Xuan Yang 2009/7/24 Zhonglin Chai My personal view on this is that for cells, we tried to avoid formation of > ice crystals which break cell membrane to kill your cells. This is achieved > by the protocol you described with slow freezing (in DMSO) and fast thawing > in 37oC degree water bath. For proteins, particularly for enzymes, or other > bioactive samples, they may be sensitive to high temperatures, the best way > to thaw is on ice to avoid exposing them to an undesirable higher > temperature such as room temperature. > > Z Chai > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu [mailto: > methods-bounces@oat.bio.indiana.edu] On Behalf Of Xuan Yang > Sent: Friday, 24 July 2009 12:25 PM > To: methods@magpie.bio.indiana.edu > Subject: Confused about the different principles to freeze & thaw cells and > purified protein > > Dear Sir or Madam, > > When we store cells (especially eukaryotic cells), storage box filled with > isopropyl alcohol was used to prevent the temperature from dropping too > fast. While thawing the cells from liquid nitrogen, we put the tube > immediately into 37?C water bath. Freezing slowly, thawing fast. This > strategy was totally contrary to the way we deal with purified proteins, > namely freezing rapidly (favorablely in liquid nitrogen), but thawing slowly > (favorablely on ice). It was quite confusing and I was wondering whether > anyone would be so kind to offer me some explanations. > > Sincerely, > > Xuan Yang > > National Laboratory of Biomacromolecules and Center for Infection and > Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, > 15 DaTun Road,Chaoyang District, Beijing, China, 100101 > Tel: 86-10-64884329 > We will either find a way or make one. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > ______________________________________________________________________ > This email has been scanned by the MessageLabs Email Security System. > ______________________________________________________________________ > > This email and any files transmitted with it are confidential and intended > solely for the use of the individual or entity to whom they are addressed. > If you have received this email in error please promptly delete this email > and notify the system manager. > > This email has been scanned by the MessageLabs Email Security System. > For more information please visit http://www.messagelabs.com/email > From MConnelly from lab901.com Fri Jul 24 05:19:25 2009 From: MConnelly from lab901.com (Matthew Connelly) Date: Fri Jul 24 07:12:46 2009 Subject: Confused about the different principles to freeze & thaw cells and purified protein In-Reply-To: References: <5A6456E670F5564EB7095EC34563B39DE4AF51@lab901-svr01.Lab901.Com> Message-ID: <5A6456E670F5564EB7095EC34563B39DE4AF8F@lab901-svr01.Lab901.Com> I don't know whether cryostability of the protein would help indicate ease of crystallisation, as you are to some extent altering the nature of the protein by crystallising it, whereas in freezing you are trying to avoid modifications to the structure for preservation purposes. However, you are right that freeze concentration and suparsaturation sound like similar processes, so there may be a corollary between protein stability on freezing and how closely the crystallised structure reflects the native protein structure. After all, both reflect the proteins tendency to resist structural changes in low water environments. Unfortunately I am no expert on protein crystallography so I have no idea how likely that is! I must say I'd never thought that cells might be easier to store frozen than proteins before, that is very interesting! From: Xuan Yang [mailto:pattisyang@gmail.com] Sent: 24 July 2009 11:05 To: Matthew Connelly Cc: methods@magpie.bio.indiana.edu Subject: Re: Confused about the different principles to freeze & thaw cells and purified protein Dear Matt, Thanks for the great & indepth explanation! You must be very good at physics. Interestingly, it was obvious that some proteins were born tougher than others and could tolerate freezing and thawing process much better. Since the freezing process seemed like a process of supersaturation, I was wondering whether a better tolerance would indicate a better chance to form crystals. Moreover, Prof. Terese M. Bergfors mentioned in the book "Protein Crystallization" that "Cells or bacteria tolerate freezing (-70?C) better than many purified proteins", as a result, it would better to store expression hosts rather than purified proteins. And since cryopreservation was cell/tissue type dependent, I was wondering whether anyone would like to share some nice strategies to store expression hosts, especially E.coli. Thanks in advance! Sincerely, Xuan Yang 2009/7/24 Matthew Connelly When you freeze aqueous solutions slowly you form larger ice crystals. This would segregate water away from proteins and into the crystals of ice. As the amount of unfrozen water drops the protein becomes more and more concentrated and loses the water shell required to maintain its structure (remember the primary force for protein structure is the organisation of hydrophobic and hydrophilic residues). This quickly leads to protein denaturation and aggregation, and is one of the reasons why freeze thaw cycles are so damaging to proteins. Proteins are frozen quickly to produce the smallest ice crystals possible as quickly as possible, which minimises the disruption described above. Formulation of the protein with excipients such as polyols and carbohydrates can also help dramatically improve stability during freeze thaw by helping to lower the total amount of water bound up in ice crystals, and to take the place of water at the proteins surface. This can allow you to freeze proteins more slowly without causing damage. Not sure about cells, but I think that the freezing rates necessary for cryopreservation are actually dependent on the cell/tissue type... Matt -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Xuan Yang Sent: 24 July 2009 03:25 To: methods@magpie.bio.indiana.edu Subject: Confused about the different principles to freeze & thaw cells and purified protein Dear Sir or Madam, When we store cells (especially eukaryotic cells), storage box filled with isopropyl alcohol was used to prevent the temperature from dropping too fast. While thawing the cells from liquid nitrogen, we put the tube immediately into 37?C water bath. Freezing slowly, thawing fast. This strategy was totally contrary to the way we deal with purified proteins, namely freezing rapidly (favorablely in liquid nitrogen), but thawing slowly (favorablely on ice). It was quite confusing and I was wondering whether anyone would be so kind to offer me some explanations. Sincerely, Xuan Yang National Laboratory of Biomacromolecules and Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617, 15 DaTun Road,Chaoyang District, Beijing, China, 100101 Tel: 86-10-64884329 We will either find a way or make one. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From pattisyang from gmail.com Fri Jul 24 05:05:00 2009 From: pattisyang from gmail.com (Xuan Yang) Date: Fri Jul 24 07:12:56 2009 Subject: Confused about the different principles to freeze & thaw cells and purified protein In-Reply-To: <5A6456E670F5564EB7095EC34563B39DE4AF51@lab901-svr01.Lab901.Com> References: <5A6456E670F5564EB7095EC34563B39DE4AF51@lab901-svr01.Lab901.Com> Message-ID: Dear Matt, Thanks for the great & indepth explanation! You must be very good at physics. Interestingly, it was obvious that some proteins were born tougher than others and could tolerate freezing and thawing process much better. Since the freezing process seemed like a process of supersaturation, I was wondering whether a better tolerance would indicate a better chance to form crystals. Moreover, Prof. Terese M. Bergfors mentioned in the book "Protein Crystallization" that "Cells or bacteria tolerate freezing (-70?C) better than many purified proteins", as a result, it would better to store expression hosts rather than purified proteins. And since cryopreservation was cell/tissue type dependent, I was wondering whether anyone would like to share some nice strategies to store expression hosts, especially E.coli. Thanks in advance! Sincerely, Xuan Yang 2009/7/24 Matthew Connelly > When you freeze aqueous solutions slowly you form larger ice crystals. This > would segregate water away from proteins and into the crystals of ice. As > the amount of unfrozen water drops the protein becomes more and more > concentrated and loses the water shell required to maintain its structure > (remember the primary force for protein structure is the organisation of > hydrophobic and hydrophilic residues). This quickly leads to protein > denaturation and aggregation, and is one of the reasons why freeze thaw > cycles are so damaging to proteins. > > Proteins are frozen quickly to produce the smallest ice crystals possible > as quickly as possible, which minimises the disruption described above. > Formulation of the protein with excipients such as polyols and carbohydrates > can also help dramatically improve stability during freeze thaw by helping > to lower the total amount of water bound up in ice crystals, and to take the > place of water at the proteins surface. This can allow you to freeze > proteins more slowly without causing damage. > > Not sure about cells, but I think that the freezing rates necessary for > cryopreservation are actually dependent on the cell/tissue type... > > Matt > > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu [mailto: > methods-bounces@oat.bio.indiana.edu] On Behalf Of Xuan Yang > Sent: 24 July 2009 03:25 > To: methods@magpie.bio.indiana.edu > Subject: Confused about the different principles to freeze & thaw cells and > purified protein > > Dear Sir or Madam, > > When we store cells (especially eukaryotic cells), storage box filled with > isopropyl alcohol was used to prevent the temperature from dropping too > fast. While thawing the cells from liquid nitrogen, we put the tube > immediately into 37?C water bath. Freezing slowly, thawing fast. This > strategy was totally contrary to the way we deal with purified proteins, > namely freezing rapidly (favorablely in liquid nitrogen), but thawing > slowly > (favorablely on ice). It was quite confusing and I was wondering whether > anyone would be so kind to offer me some explanations. > > Sincerely, > > Xuan Yang > > National Laboratory of Biomacromolecules and > Center for Infection and Immunity, > Institute of Biophysics, > Chinese Academy of Sciences, > Room 1617, 15 DaTun Road,Chaoyang District, > Beijing, China, 100101 > Tel: 86-10-64884329 > We will either find a way or make one. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From allison from nospam.com Fri Jul 24 08:57:07 2009 From: allison from nospam.com (Allison) Date: Fri Jul 24 11:04:07 2009 Subject: can protein modifications affect immunodetection? In-Reply-To: <5b6700d8-7e4c-422d-8673-dbba14a7cbad@h11g2000yqb.googlegroups.com> References: <5b6700d8-7e4c-422d-8673-dbba14a7cbad@h11g2000yqb.googlegroups.com> Message-ID: <4a69bd62$0$19647$9a6e19ea@news.newshosting.com> Scientist wrote: > Hi all! > im trying to detect a protein in plants Crude extract using > polyclonal Abs. These Abs were obtained from rat sorum, The protein > used to innolculate the rats was expressed in E.coli (thus lacking the > typical postrasductional modifications occurring in plants) . > Untill only no positve results were obtained. > When recombinant form is used as positive control everthing works > well. > So the possibilities are : > a) native form is absent in the plant. > b) the protien is present but in low concetration > c) native form is modified in someway , thus the abs cannot recognize > the epitopes. > > have anyone a suggestions for these items? > thanks a lot Try immunizing more than one rat - it's possible that different animals will respond differently to the same antigen. I'm in the middle of a project where only one of five mice pick up the endogenous mammalian protein on Western after being immunized with the recombinant protein. Allisoin From yvonne.couch from pharm.ox.ac.uk Mon Jul 27 05:23:35 2009 From: yvonne.couch from pharm.ox.ac.uk (Yvonne Couch) Date: Mon Jul 27 11:44:30 2009 Subject: Endotoxins Message-ID: <003d01ca0ea4$4cb22850$e61678f0$@couch@pharm.ox.ac.uk> Hi all, If I HCl purify a peptide made in E.Coli, is there likely to be a large amount of endotoxic material left in the final solution and if so is it possible to get rid of this? Cheers Yvonne From editor from gene-quantification.info Mon Jul 27 02:24:06 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Mon Jul 27 11:52:15 2009 Subject: qPCR NEWS July 2009 - focus on single-cell qPCR Message-ID: <11590046-c962-4333-862f-a0f291a19e89@g31g2000yqc.googlegroups.com> qPCR NEWS July 2009 - focus on single-cell qPCR ----------------------------------------------------------------- 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 single-cell real-time PCR - qPCR / real-time PCR BLOG => http://real-time-pcr.blogspot.com/ - qPCR LinkedIN group - real-time PCR & qPCR => http://www.linkedin.com/groups?about=&gid=149835&trk=anet_ug_grppro - For better navigation we developed a TAG CLOUD => http://directory.gene-quantification.info/ - New qPCR events in autumn 2009: symposia & workshops => http://events.gene-quantification.info/ European wide qPCR application workshops => register now ! => course program spring - summer - autumn 2009 => http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf => http://www.gene-quantification.de/single-cell-qpcr-course-sept-2009.pdf -------------------------------------------------------------------------------- Single-cell molecular-biology is a relatively new scientific branch in biology. The first single-cell analysis were involved in the characterization of mitochondrial DNA in 1988. Single-cell DNA analysis, in particular genomic DNA, is important and may be informative in the analysis of genetics of cell clonality, genetic anticipation and single-cell DNA polymorphisms. Nowadays for most scientists the quantitative transcriptomics in a single-cell is much more important, and the analytical method of choice is the quantitative real-time RT-PCR. The relative abundance of single mRNAs and their up- or down-regulation in a single cell, compared to their neighbour cells, is the goal. The need for quantitative single-cell mRNA analysis is evident given the vast cellular heterogeneity of all tissue cells and the inability of conventional RNA methods, like northern blotting, RNAse protection assay or classical block RT-PCR, to distinguish individual cellular contributions to mRNA abundance differences. => http://singlecell.gene-quantification.info/ -------------------------------------------------------------------------------- new papers => http://www.gene-quantification.de/singlecell3.html - Imaging intracellular RNA distribution and dynamics in living cells. - mRNA-Seq whole-transcriptome analysis of a single cell. - Genomic expression analysis by single-cell mRNA differential display of quiescent CD8 T cells from tumour-infiltrating lymphocytes obtained from in vivo liver tumours. - Rac1 regulates pancreatic islet morphogenesis. - A novel single-cell quantitative real-time RT-PCR method for quantifying foot-and-mouth disease viral RNA. - Quantification of circulating endothelial and progenitor cells: comparison of quantitative PCR and four-channel flow cytometry. - Prognosis of non-small cell lung cancer patients by detecting circulating cancer cells in the peripheral blood with multiple marker genes. - Nanoliter reactors improve multiple displacement amplification of genomes from single cells. - Fluidigm Dynamic Arrays provide a platform for single-cell gene expression analysis. - Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte. - Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. - Intracellular Gene Expression Profi les Revealed with Real-time PCR Tomography -------------------------------------------------------------------------------- NEW blogs & groups qPCR / real-time PCR BLOG => http://real-time-pcr.blogspot.com/ qPCR LinkedIN group - real-time PCR & qPCR => http://www.linkedin.com/groups?about=&gid=149835&trk=anet_ug_grppro This group wants to join worldwide users in the field of real-time quantitative PCR. It offers a forum where new ideas can be exchanged, and allows networking with other people in this field. -------------------------------------------------------------------------------- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, etc. Please submit your qPCR event here => events@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.) => http://site.bioeps.com/index.php?option=com_seminar&Itemid=6 14 - 16 September 2009 (E) NEW single-cell qPCR 19 - 23 September 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 26 - 28 October 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) 16 - 20 November 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) 7 - 11 December 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics (Thu. - Fri.) => http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf => http://www.gene-quantification.de/single-cell-qpcr-course-sept-2009.pdf -------------------------------------------------------------------------------- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info -------------------------------------------------------------------------------- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright ? 2005 - 2009 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com 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 briancady413 from yahoo.com Tue Jul 28 10:31:18 2009 From: briancady413 from yahoo.com (briancady413) Date: Tue Jul 28 11:15:45 2009 Subject: EDTA solution becomes slurry References: Message-ID: <94c1928d-89f3-4203-9845-6754d3efbe27@l34g2000vba.googlegroups.com> On May 31, 5:41?pm, Tom Knight wrote: >...?Just a reminder, you cannot make a 1 M > solution -- the stock solution is 500 mM. Tom, is there a reference for this? A colleague is attempting to make 1M EDTA, with great difficulty. I guess I will search for solubility info. Thanks in advance, Brian Cady From yasirphr from yahoo.com Tue Jul 28 08:49:52 2009 From: yasirphr from yahoo.com (muhammad yasir) Date: Tue Jul 28 11:15:53 2009 Subject: storing of enzyme Message-ID: <518007.2488.qm@web56007.mail.re3.yahoo.com> I have purified cellulase enzyme through His tage and interested to store the purified protein. I shall be glad to know the recepi for stable storge of purified enzyme. From agbiok4 from gmail.com Tue Jul 28 04:45:11 2009 From: agbiok4 from gmail.com (kamalaker nasani) Date: Tue Jul 28 11:15:59 2009 Subject: AGROBACTERIUM Message-ID: I need small information. I have to prepare Glycerol permanents of my strains. Please anybody can tell the concentration of antibitics and what antibiotics need to be used for EHA101, EHA105 and LBA4404. -- Kamalaker Nasani, Biotechnologist, Global Transgenes Ltd. From hroychow from nmsu.edu Tue Jul 28 14:51:26 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Tue Jul 28 15:46:32 2009 Subject: storing of enzyme In-Reply-To: <518007.2488.qm@web56007.mail.re3.yahoo.com> References: <518007.2488.qm@web56007.mail.re3.yahoo.com> Message-ID: <2193.128.123.174.0.1248810686.squirrel@webmail.nmsu.edu> Use a buffer (10mM Tris-EDTA, pH 6.5 to 7.2, or 50mM phosphate buffer should work) and add glycerol to 35% (as high as 50%). Store in a -20C freezer (no auto defrost). Cellulase can also be stores freeze dried after the appropriate dialysis against distilled water containing 1mM DTT. > > I have purified cellulase enzyme through His tage and interested to store > the purified protein. I shall be glad to know the recepi for stable storge > of purified enzyme. > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From nick.theodorakis from gmail.com Tue Jul 28 16:55:30 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Tue Jul 28 21:58:41 2009 Subject: storing of enzyme References: <518007.2488.qm@web56007.mail.re3.yahoo.com> Message-ID: On Jul 28, 3:51?pm, "Dr. Hiranya S. Roychowdhury" wrote: > Use a buffer (10mM Tris-EDTA, pH 6.5 to 7.2, or 50mM phosphate buffer > should work) and add glycerol to 35% (as high as 50%). ?Store in a -20C > freezer (no auto defrost). > > Cellulase can also be stores freeze dried after the appropriate dialysis > against distilled water containing 1mM DTT. > Is dialysis tubing still made from cellulose? Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From cooperl from onid.orst.edu Tue Jul 28 17:25:28 2009 From: cooperl from onid.orst.edu (Laurel Cooper) Date: Tue Jul 28 21:58:48 2009 Subject: guanidinium disposal Message-ID: <006901ca0fd2$4fcca180$ef65e480$@orst.edu> I am cleaning out a bunch of old stuff in the lab I am in and there are numerous bottles of guanidinium isothiocyanate and guanidinium chloride solutions. Can these be disposed of down the drain with lots of water or do they need to go to waste disposal. They are solutions from commercial kits. Thanks From hroychow from nmsu.edu Wed Jul 29 10:26:15 2009 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Wed Jul 29 12:27:09 2009 Subject: storing of enzyme In-Reply-To: References: <518007.2488.qm@web56007.mail.re3.yahoo.com> Message-ID: <1603.128.123.174.0.1248881175.squirrel@webmail.nmsu.edu> Mostly cellulose nitrate. I had done it "way back when ..." using these. > On Jul 28, 3:51?pm, "Dr. Hiranya S. Roychowdhury" > wrote: >> Use a buffer (10mM Tris-EDTA, pH 6.5 to 7.2, or 50mM phosphate buffer >> should work) and add glycerol to 35% (as high as 50%). ?Store in a -20C >> freezer (no auto defrost). >> >> Cellulase can also be stores freeze dried after the appropriate dialysis >> against distilled water containing 1mM DTT. >> > > Is dialysis tubing still made from cellulose? > > Nick > > -- > Nick Theodorakis > nick_theodorakis@hotmail.com > contact form: > http://theodorakis.net/contact.html > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From camjay from gmail.com Wed Jul 29 10:26:38 2009 From: camjay from gmail.com (camjay) Date: Wed Jul 29 12:27:16 2009 Subject: guanidinium disposal References: Message-ID: On 28 July, 23:25, "Laurel Cooper" wrote: > I am cleaning out a bunch of old stuff in the lab I am in and there are > numerous bottles of guanidinium isothiocyanate and guanidinium chloride > solutions. ?Can these be disposed of down the drain with lots of water or do > they need to go to waste disposal. ?They are solutions from commercial kits. Here in Europe guanidinium isothiocyanate is rated "Harmful to aquatic organisms" and "May cause long-term adverse effects in the aquatic environment" and I certainly wouldn't dispose of any down the drain. Really you should seek local guidance from your institute, water supply or sewerage company or appropriate regulatory authority. From sudhee26 from gmail.com Wed Jul 29 14:07:45 2009 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Wed Jul 29 16:45:27 2009 Subject: need to identify a contaminant in the cell culture Message-ID: Hi, all,I recently found this contaminant in one of the thawed vials..i want to ID it.i dont know how to post pictures in this list..but i will give description.. 1. macroscopically: it appears floating mass of myecilium: two latyers..layer 1-close to cell surface sometimes sticking to cells..layer two: surface of media..looks like oil has spilled in media..looks greasy..very difficult to get rid of even after repeated PBS washings..layer 1: is clump of mycelia..layer 2: looks like broken mycelia floating..both layer1 and layer 2 are not motile. 2. cell morphology does not seem to change a lot..cells look healthy.but they die suddenly when the contaminant number flourishes. no change in media color except media top looks greasy/oily..imagine oil spilled in sea!!!..they grow in FBS+pen-strep, they are resistant to geneticin too.. 3. i took the supernatant and plated on LB agar (thats the only thing i have--i dont do microbiology :))..with ampicillin (since i found out that this is penicillin resistant)..within 6 days i small colonies which increased up to 3 mm diameter by 15 days..all colonies have same morphology..i guess hence its not something spurious..and also on two different plates i got same kind of colonies. 4. Form: Circular (grossly, but when looked at 10X magnification we can make out that its not so) Color: Cream Margin: undulating Elevation: umbonate Surface: smooth. Macroscopically surface looks infrequently studdent with cream colored spekles. Opacity: Opaque 5. 40X magnification revealed very interestingly margin of this colony which appeared on LB plate with ampicillin had Claw like projection around..covering almost entire periphery (around) of the colony..probably thats how colony spreads and becomes big..also at 40X the colony does not look homogenous..its made of fine grains.. so what is it..I will try posting some pics to my blog.. meanwhile..i really appreciate the ID..so that I can get rid of it..i am anyway ordering amphotericin B. regards sudheendra. -- Think before agree Think before you nod STOP thinking to be a God From markydor from yahoo.com.br Wed Jul 29 18:49:14 2009 From: markydor from yahoo.com.br (Marcos Moraes) Date: Wed Jul 29 22:09:03 2009 Subject: About SSCP Message-ID: <204388.64103.qm@web110313.mail.gq1.yahoo.com> Dear Bruno, Surfing on net?I found a message (http://www.bio.net/bionet/mm/methods/2002-February/092258.html) that you?said things about?SSCP resolution. In that moment, you said you was running?1XTBE?+ 10% glycerol gels.?How many conditions have you ever tested? I'm asking because after a long time screening for mutation for?COL1A1 gene (Osteogenesis imperfecta), i realized that the low ratio of mutants could be the SSCP conditions. Here,?the SSCP runs at room temperature (PAGE-SSCP S2 Biometra) and at the moment, I'm trying to figure out a condition(s) capable to detect the mutations. I'm testing absence, 5% and 10% glycerol; no glycerol + 10% sucrose; MDE 0.5X?w/ or w/out glycerol 5%, all of them with 0.5X TBE. Do you would recommend me to use 1X TBE instead of 0.5X? Does it make any difference for detection? Best regards, ? Marcos?Moraes| Mestrando em Biotecnologia |?Sa?de Laborat?rio de?Gen?tica Humana e Molecular ? N?cleo de Gen?tica Humana e Molecular |?Depto.?Ci?ncias Biol?gicas |?Centro de Ci?ncias Humanas e Naturais | Universidade Federal do Esp?rito Santo Av. Marechal Campos,?1468 - Maru?pe -?29040-090 - Vit?ria - ES - Brasil T?+55 27 3335 7253 |?C+55 27 9249 0190 |?markydor@yahoo.com.br http://lattes.cnpq.br/0799432282422121 ____________________________________________________________________________________ Veja quais s?o os assuntos do momento no Yahoo! +Buscados http://br.maisbuscados.yahoo.com From C.P.Pena-Diaz from 2006.hull.ac.uk Thu Jul 30 10:47:14 2009 From: C.P.Pena-Diaz from 2006.hull.ac.uk (Carmen P Pena Diaz) Date: Thu Jul 30 11:08:48 2009 Subject: reconstitution of membrane proteins Message-ID: <445800D8FD5D3D43878FD7D2842A304743542C@EXCL2VS2.adir.hull.ac.uk> Hello I was wondering how many of you work with membrane proteins and might me able to help out with this one. I'm trying to reconstitute mitochondrial carriers into liposomes. The established protocol is done in 3 major steps: a) isolating the protein, of course, from bacterial strain after expression. the isolation is basically from inclusion bodies and solubilization in 1% sarkosyl in tris buffer. b) making the proteoliposomes, which is done incorporating the protein in extruded egg yolk phospholipids (9mg/mL), internal substrate (ADP or ATP in my case, 30mM), Tx-114 and cardiolipin in 10mM buffer MOPS, 50mM NaCl. this mix is passed through a Amberlite XAD-2 column, 24X the un-incorporated internal substrate is eliminated using another column, Dowex AG1-X8, equilibrated with the same buffer MOPS as before c) the transport assay is done mixing the liposomes with ATP (3H) and stoping the reaction with 30mM pyridoxal 5? phosphate and 10mM bathophenanthroline after 0,2,5 & 10minutes. excess of non-trasported radiactivity is eliminated using sephadex G-75 equilibrated with the same Mops buffer as before. So far, no transport has been observed. I was wondering if any of you could shed some light on the subject. regards Priscila -------------- next part -------------- ***************************************************************************************** To view the terms under which this email is distributed, please go to http://www.hull.ac.uk/legal/email_disclaimer.html ***************************************************************************************** From glenn.dunshea from gmail.com Thu Jul 30 00:34:45 2009 From: glenn.dunshea from gmail.com (Glenn Dunshea) Date: Thu Jul 30 11:08:57 2009 Subject: About SSCP In-Reply-To: <204388.64103.qm@web110313.mail.gq1.yahoo.com> References: <204388.64103.qm@web110313.mail.gq1.yahoo.com> Message-ID: <8acca5110907292234n192e2404t1585c53ce58d2b28@mail.gmail.com> I have found MDE solution excellent for detection of mutations without the need for glycerol. Something to consider when running your trials is that one of the most critical parameters for SSCP assays is constant temperature. I would not recommend running SSCP at room temperature.. I hope your experiments are successful. Cheers, Glenn On Thu, Jul 30, 2009 at 7:49 AM, Marcos Moraes wrote: > Dear Bruno, > > Surfing on net I found a message ( > http://www.bio.net/bionet/mm/methods/2002-February/092258.html) that > you said things about SSCP resolution. In that moment, you said you was > running 1XTBE + 10% glycerol gels. How many conditions have you ever tested? > I'm asking because after a long time screening for mutation for COL1A1 gene > (Osteogenesis imperfecta), i realized that the low ratio of mutants could be > the SSCP conditions. Here, the SSCP runs at room temperature (PAGE-SSCP S2 > Biometra) and at the moment, I'm trying to figure out a condition(s) capable > to detect the mutations. I'm testing absence, 5% and 10% glycerol; no > glycerol + 10% sucrose; MDE 0.5X w/ or w/out glycerol 5%, all of them with > 0.5X TBE. Do you would recommend me to use 1X TBE instead of 0.5X? Does it > make any difference for detection? > > Best regards, > > Marcos Moraes| Mestrando em Biotecnologia | Sa?de > Laborat?rio de Gen?tica Humana e Molecular > > N?cleo de Gen?tica Humana e Molecular | Depto. Ci?ncias Biol?gicas | Centro > de Ci?ncias Humanas e Naturais | Universidade Federal do Esp?rito Santo > Av. Marechal Campos, 1468 - Maru?pe - 29040-090 - Vit?ria - ES - Brasil > T +55 27 3335 7253 | C+55 27 9249 0190 | markydor@yahoo.com.br > http://lattes.cnpq.br/0799432282422121 > > > > ____________________________________________________________________________________ > 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 contreras from fmed.edu.uy Thu Jul 30 09:48:29 2009 From: contreras from fmed.edu.uy (Paola Contreras) Date: Thu Jul 30 11:09:13 2009 Subject: phmeter Message-ID: <9df709230907300748ra724e4j77176a1fb3b87b7e@mail.gmail.com> I found in the methods mailing list the following: We bought a Sentron model 1001. that is why I thought you can help me since in our lab we have the same phmeter but without the corresponding instructions for users. I would appreciate if someone could give me an idea on how to get the users' manual. I have already asked the makers but did not get an answer. best regards, Paola. -- Paola Contreras, MSc Departamento de Fisiolog?a Facultad de Medicina Universidad de la Rep?blica Montevideo, URUGUAY Tel: 598 2 924 94 53 From sudhee26 from gmail.com Thu Jul 30 12:33:10 2009 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu Jul 30 14:54:11 2009 Subject: need to identify a contaminant in the cell culture In-Reply-To: <20090730165021.27284angk80bt3s4@webmail.irb.hr> References: <20090730165021.27284angk80bt3s4@webmail.irb.hr> Message-ID: @ Val: Yup, doing that. getting rid of contaminated cell vials and also doing routine stuff with incubators. Have ordered amphotericin B..I thought ID would help to combat this pain in a more precise way.. @ Jasenka: Colonies look soft and are soft. Do they dont seem to grow into agar rather they spread on the surface. I again want to mention the claw like projections from the margin of colonies..like flares from sun! i have no idea whether this description can help ID it..but thats what it is. more ID tips are eagerly awaited. regards sudheendra. On Thu, Jul 30, 2009 at 8:20 PM, Jasenka Pigac wrote: > It is hard to tell from your description, I aminclined to think about > actinomycetes, due to a rather slow growth and resistancy. Are the colonies > soft or like leather? Do they grow into agar or stay on its surface? > > Hi, all,I recently found this contaminant in one of the thawed vials..i >> want >> to ID it.i dont know how to post pictures in this list..but i will give >> description.. >> >> 1. macroscopically: it appears floating mass of myecilium: two >> latyers..layer 1-close to cell surface sometimes sticking to cells..layer >> two: surface of media..looks like oil has spilled in media..looks >> greasy..very difficult to get rid of even after repeated PBS >> washings..layer >> 1: is clump of mycelia..layer 2: looks like broken mycelia floating..both >> layer1 and layer 2 are not motile. >> >> 2. cell morphology does not seem to change a lot..cells look healthy.but >> they die suddenly when the contaminant number flourishes. no change in >> media >> color except media top looks greasy/oily..imagine oil spilled in >> sea!!!..they grow in FBS+pen-strep, they are resistant to geneticin too.. >> >> 3. i took the supernatant and plated on LB agar (thats the only thing i >> have--i dont do microbiology :))..with ampicillin (since i found out that >> this is penicillin resistant)..within 6 days i small colonies which >> increased up to 3 mm diameter by 15 days..all colonies have same >> morphology..i guess hence its not something spurious..and also on two >> different plates i got same kind of colonies. >> >> 4. >> Form: Circular (grossly, but when looked at 10X magnification we can make >> out that its not so) >> Color: Cream >> Margin: undulating >> Elevation: umbonate >> Surface: smooth. Macroscopically surface looks infrequently studdent with >> cream colored spekles. >> Opacity: Opaque >> >> 5. 40X magnification revealed very interestingly margin of this colony >> which >> appeared on LB plate with ampicillin had Claw like projection >> around..covering almost entire periphery (around) of the colony..probably >> thats how colony spreads and becomes big..also at 40X the colony does not >> look homogenous..its made of fine grains.. >> >> so what is it..I will try posting some pics to my blog.. >> >> meanwhile..i really appreciate the ID..so that I can get rid of it..i am >> anyway ordering amphotericin B. >> >> regards >> sudheendra. >> >> >> >> -- >> Think before agree >> Think before you nod >> STOP thinking >> to be a God >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> >> > > -- Think before agree Think before you nod STOP thinking to be a God