From sumida from bbri.org Fri Jul 4 02:30:12 2008 From: sumida from bbri.org (jps) Date: Fri Jul 4 10:43:22 2008 Subject: [Protein-analysis] preparing proteins for lyphilization Message-ID: <279a3698-1bd7-4ec3-8887-62e9efc4f4d4@t12g2000prg.googlegroups.com> I think it is common practice to dialyze proteins in an ammonium bicarbonate solution at pH=8. Presumably this is a way to maintain the protein with some salt to stabilize it while in solution, but not have any salt present after this protein solution is lyophilized since the ammonia sublimates and carbon dioxide is released during freeze drying step. However, I am wondering if this is actually accurate. Can anybody comment on this procedure? From ozgun.harmanci from gmail.com Thu Jul 3 23:56:29 2008 From: ozgun.harmanci from gmail.com (ozgun.harmanci) Date: Fri Jul 4 10:45:24 2008 Subject: [Protein-analysis] missing residues in pdb file Message-ID: Hello, I am having trouuble with missing residues in pdb files and need to replace them (i.e. interpolate them) using a program. I think swiss- pdbviewer and pymol do that. My problem is that I am not very capable of using those programs and the person whom I asked for help told me that I have to check for missing residues in pdb file, open swiss- pdbviewer then add residue at correct place and finally she told me that I might need to do an energy minimization and I am thinking that I will definitely do a mistake there. I was wondering if anyone knows a more automated way to get the missing residues inserted for me because I am thinking that you can find missing residues in pdb and and programs can read that info and add these missing residues themselves automatically.. (but again maybe not..) Thanks. Sincerely, Arif. From sumida from bbri.org Fri Jul 11 21:06:57 2008 From: sumida from bbri.org (jps) Date: Sat Jul 12 11:54:11 2008 Subject: [Protein-analysis] Re: preparing proteins for lyphilization References: <279a3698-1bd7-4ec3-8887-62e9efc4f4d4@t12g2000prg.googlegroups.com> Message-ID: <4033f296-a7df-4e2f-b679-64375d20a8b3@56g2000hsm.googlegroups.com> Thank you for your response to my post. I have found this to be generally true, and I remember that it was told to me sometime ago, but there are some questions I wonder about. One would think that the pH could drop significantly during this process; I normally use a 5 to 10mM buffer concentration which would not have a significant buffering capacity. I suppose the reasoning is that HCO3 would eventually convert to CO2 and be lost. But what happens to the solution freezing point and consequently the vapor pressure. Wouldn't it become more and more difficult to sublimate the dissolved gases as the solute concentration increases? I cannot seem to find any type of literature reference for this piece of information. Do you know if anyone has performed any physical characterization of this process? I have been searching pubmed and JSTOR but have yet to find anything. Best regards. JPS On Jul 7, 11:59?am, d...@no.email.thankstospam.net (DK) wrote: > In article <279a3698-1bd7-4ec3-8887-62e9efc4f...@t12g2000prg.googlegroups.com>, jps wrote: > > >I think it is common practice to dialyze proteins in an ammonium > >bicarbonate solution at pH=8. ?Presumably this is a way to maintain > >the protein with some salt to stabilize it while in solution, but not > >have any salt present after this protein solution is lyophilized since > >the ammonia sublimates and carbon dioxide is released during freeze > >drying step. > > >However, I am wondering if this is actually accurate. ?Can anybody > >comment on this procedure? > > This is actually accurate. Of course, some proteins survive lyo just > fine while others tend to die. You never know until you try. Addding > some sugars into protein solution to be lyophilized frequently helps > because they act as cryoprotectants. Trehalose is very popular but > sucrose is always worth trying (trehalose is usually less pure). > > DK From sumida from bbri.org Fri Jul 11 21:11:37 2008 From: sumida from bbri.org (jps) Date: Sat Jul 12 11:54:17 2008 Subject: [Protein-analysis] NEM-S1 Message-ID: N-ethylmaleimide modification of the SH1 and SH2 groups appears to be a frequently used method to convert the myosin S1 subunit into a strong-binding but inactive (ATPase-wise) form of the S1. This seems to have been a useful tool to investigate the regulatory properties of the muscle thin filament activation and cooperativity. Can anyone comment on the modification process? Is the actin/NEM-S1 binding constant well characterized? Is it always the same? Does NEM- S1 precipitate out of solution over time? Thank you. From dorit.grunberger from radiology.ucsf.edu Mon Jul 14 20:33:40 2008 From: dorit.grunberger from radiology.ucsf.edu (Dorit Grunberger) Date: Mon Jul 14 21:07:26 2008 Subject: [Protein-analysis] Freeze Dryer vs Speed Vac Message-ID: Saw this online and thought to ask you if it makes sense to hook up a speedvac to a lyophilizer both as source of vacuum and no less important, a cold environment to keep my extract happy. I?m doing chloroform : methanol : water extracts of small tissue samples. The total volumes for extraction will be small and I was hoping to use a speedvac to dry/concentrate my MeOH/H2O phase. It?s critical for the sample to remain as cold as possible, since the analysis of contents is done by NMR and we?re hoping to maintain integrity of some very easily degradable metabolites (phospho-choline for example). Bottom line: do you think this will work? Thanks in advance for any help. Dorit Grunberger Department of Radiology email: dorit.grunberger@radiology.ucsf.edu 415.514.4845 office 415.385.0977 cell 415.514.2550 fax From sumida from bbri.org Tue Jul 15 09:24:53 2008 From: sumida from bbri.org (jps) Date: Tue Jul 15 13:12:43 2008 Subject: [Protein-analysis] Re: Freeze Dryer vs Speed Vac References: Message-ID: I have not done what you have described. I think it would probably work. However, I'd be worried about the temperature of the sample in the speed vac, some older units I've worked with tend to let the sample get fairly warm ~30oC, and I wonder if it would be any more efficient than just using the lyophilizer as is - no speed vac. One could set up an dry-ice/acetone bath and place a liquid nitrogen cold trap between the frozen sample, maintained in the bath, and vacuum pump, but that would also be equivalent to the lyophylizer plus the time it would take to hunt down all the glassware, dry ice and acetone for the task - not to mention the waste handling. That's my 2cents. Good luck. JPS On Jul 14, 9:33?pm, Dorit Grunberger wrote: > Saw this online and thought to ask you if it makes sense to hook up a > speedvac to a lyophilizer both as source of vacuum and no less important, a > cold environment to keep my extract happy. I?m doing chloroform : methanol : > water extracts of small tissue samples. The total volumes for extraction > will be small and I was hoping to use a speedvac to dry/concentrate my > MeOH/H2O phase. It?s critical for the sample to remain as cold as possible, > since the analysis of contents is done by NMR and we?re hoping to maintain > integrity of some very easily degradable metabolites (phospho-choline for > example). > > Bottom line: do you think this will work? > > Thanks in advance for any help. > > Dorit Grunberger > Department of Radiology > email: ?dorit.grunber...@radiology.ucsf.edu > 415.514.4845 ? office > 415.385.0977 ?cell > 415.514.2550 ?fax From engelbert_buxbaum from hotmail.com Wed Jul 16 10:49:05 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jul 16 13:16:24 2008 Subject: [Protein-analysis] Re: Freeze Dryer vs Speed Vac References: Message-ID: Am 14.07.2008, 21:33 Uhr, schrieb Dorit Grunberger : > Saw this online and thought to ask you if it makes sense to hook up a > speedvac to a lyophilizer both as source of vacuum and no less > important, a > cold environment to keep my extract happy. I?m doing chloroform : > methanol : > water extracts of small tissue samples. The total volumes for extraction > will be small and I was hoping to use a speedvac to dry/concentrate my > MeOH/H2O phase. It?s critical for the sample to remain as cold as > possible, > since the analysis of contents is done by NMR and we?re hoping to > maintain > integrity of some very easily degradable metabolites (phospho-choline for > example). The lyophiliser keeps samples cold by the heat of evaporation that is constantly removed from the system. As long as your vacuum is strong enough and the solvent has not evaporated the sample will be at a temperature well below its freezing point. If there is a problem you recognise that by your sample melting. In the SpeedVac the lyophilisation process is speeded up by gently heating the sample to replace the heat of evaporation, the sample will not be quite as cold as in a lyophiliser. In addition the residue will be in a small spot due to the centrifugal force, it will be easier to take it up in a small volume later. In addition, loss of residue will be smaller (important e.g. for radioactive samples). In either case sensitive samples have to be removed immediatly after complete drying, as they will warm to room temperature (or to operating temperture in the SpeedVac) once no solvent evaporates anymore. From allison from nospam.com Wed Jul 16 11:19:23 2008 From: allison from nospam.com (allisonh) Date: Wed Jul 16 13:16:30 2008 Subject: [Protein-analysis] Re: Freeze Dryer vs Speed Vac In-Reply-To: References: Message-ID: <487e18a7$0$25318$9a6e19ea@news.newshosting.com> If a cold temperature is important maybe you could just run the Speedvac in a cold room (with the heater turned off of course). Allison Dorit Grunberger wrote: > Saw this online and thought to ask you if it makes sense to hook up a > speedvac to a lyophilizer both as source of vacuum and no less important, a > cold environment to keep my extract happy. I?m doing chloroform : methanol : > water extracts of small tissue samples. The total volumes for extraction > will be small and I was hoping to use a speedvac to dry/concentrate my > MeOH/H2O phase. It?s critical for the sample to remain as cold as possible, > since the analysis of contents is done by NMR and we?re hoping to maintain > integrity of some very easily degradable metabolites (phospho-choline for > example). > > Bottom line: do you think this will work? > > Thanks in advance for any help. > > Dorit Grunberger > Department of Radiology > email: dorit.grunberger@radiology.ucsf.edu > 415.514.4845 office > 415.385.0977 cell > 415.514.2550 fax > > > From sumida from bbri.org Thu Jul 17 09:12:58 2008 From: sumida from bbri.org (jps) Date: Thu Jul 17 12:21:24 2008 Subject: [Protein-analysis] Re: Freeze Dryer vs Speed Vac References: <487e18a7$0$25318$9a6e19ea@news.newshosting.com> Message-ID: <8ed81a54-bfb1-431c-bba0-80e31cd10d46@e53g2000hsa.googlegroups.com> On Jul 16, 12:19?pm, allisonh wrote: > If a cold temperature is important maybe you could just run the Speedvac > in a cold room (with the heater turned off of course). > Allison > > > > Dorit Grunberger wrote: > > Saw this online and thought to ask you if it makes sense to hook up a > > speedvac to a lyophilizer both as source of vacuum and no less important, a > > cold environment to keep my extract happy. I?m doing chloroform : methanol : > > water extracts of small tissue samples. The total volumes for extraction > > will be small and I was hoping to use a speedvac to dry/concentrate my > > MeOH/H2O phase. It?s critical for the sample to remain as cold as possible, > > since the analysis of contents is done by NMR and we?re hoping to maintain > > integrity of some very easily degradable metabolites (phospho-choline for > > example). > > > Bottom line: do you think this will work? > > > Thanks in advance for any help. > > > Dorit Grunberger > > Department of Radiology > > email: ?dorit.grunber...@radiology.ucsf.edu > > 415.514.4845 ? office > > 415.385.0977 ?cell > > 415.514.2550 ?fax- Hide quoted text - > > - Show quoted text - Here's another question along these lines: canthe lyophylization process itself does not denature your protein? From sticher from bioc.unizh.ch Tue Jul 22 04:33:44 2008 From: sticher from bioc.unizh.ch (Patrick Sticher) Date: Tue Jul 22 19:21:34 2008 Subject: [Protein-analysis] 6th NCCR Symposium New Trends in Structural Biology - Program online Message-ID: <4885A978.4040107@bioc.unizh.ch> Dear colleagues, 6th International NCCR Symposium on New Trends in Structural Biology 8 + 9 September 2008, University of Z?rich, Lecture Hall KOH-B10, Z?rich, Switzerland www.structuralbiology.uzh.ch/symposium2008.asp The lecture program is available online at: http://www.structuralbiology.uzh.ch/symposium2008_lectures.asp Plenary lecturers: Markus G. Gr?tter, Stephen C. Kowalczykowski, Kaspar Locher, Keiichi Namba, Poul Nissen, Andrej Sali, Ilme Schlichting, Titia Sixma, Jeffrey Skolnick, A. Joshua Wand The meeting will be held together with the Annual Meeting of the Swiss Crystallography Society. Society lectures will be given by Clemens Schulze-Briese and Colin Nave Online registration to this event is still possible through the symposium homepage or directly at: http://www.structuralbiology.uzh.ch/registration08.asp Please do not hesitate to contact me anytime if you need further information (sticher@bioc.uzh.ch). With best regards, Patrick Sticher The NCCR Structural Biology is a research initiative of the Swiss Science Foundation. Its research encompasses the fields of recombinant protein technologies, macromolecular structure determination and computational biomolecular sciences with a special focus on membrane proteins and supramolecular assemblies/interactions. 19 research groups from Swiss Universities and Research Institutions participate in this network. www.structuralbiology.uzh.ch/ _________________________________ Dr. Patrick Sticher Moser NCCR Scientific Officer Institute of Biochemistry University of Z?rich Winterthurerstrasse 190 CH - 8057 Z?rich From engelbert_buxbaum from hotmail.com Sun Jul 27 14:35:15 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Jul 28 12:45:28 2008 Subject: [Protein-analysis] Re: Freeze Dryer vs Speed Vac References: <487e18a7$0$25318$9a6e19ea@news.newshosting.com> <8ed81a54-bfb1-431c-bba0-80e31cd10d46@e53g2000hsa.googlegroups.com> Message-ID: Am 17.07.2008, 10:12 Uhr, schrieb jps : > Here's another question along these lines: canthe lyophylization > process itself does not denature your protein? Potentially yes, that depends on the protein and the exact conditions used. Companies have entire departments that develope suitable protocols for lyophilising and reconstituting proteins of interest. This is black art, not science. Sometimes addition of polyols like trehalose helps.