Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
I also recommend you to check Dan's link on the paper for stabilising protein solubility. I have similar experience. I got high yield, soluble protein but aggregates once I have purified concentrated protein. Before venturing to the detergent area, I would suggest using other additives first (kosmotropes, chaotropes, amino acid, sugars and alcohols). I managed to get it work in high NaCl, KCl and NaBr. I managed to purify the protein, put it in Tris buffer with low NaCl. I concentrated the protein and work fast enough before it crashes (my protein usually crashes overnight). Then aliquot out the protein in different eppendorf tubes containing different additives, leave it overnight; the next day, I spun all the tubes and check for aggregation (of course, make sure you leave a control, which in my case is Tris buffer, low NaCl; just for a point of comparison). I got 7 tubes then which do not have aggregated pellet; I left them for few more days; 4 of them aggregated, and 3 of them looks okay. Allan Quoting Vitali Stanevich stanev...@wisc.edu: Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali -- Allan Pang Visiting Scientist G35 Joseph Priestley Building Queen Mary University of London 327 Mile End Road London, UK E1 4NS Phone number: 02078828480 Twitter: @xerophytes
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Going in a different direction with my reply here. Is your pI close to your current buffer ? Move at least one unit away from the theoretical pI. Do you have a friend with a real time pcr machine ? Then get some sypro orange and check out the thermal stability of your protein under various conditions. Google for thermal melt Ericsson or Greg Crowther you'll get a paper (actually these are two different papers). Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://lupo.jhsph.edu On Oct 12, 2012, at 12:52, Vitali Stanevich stanev...@wisc.edu wrote: Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
[ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Hi, Yes, it is worth trying. Nonionic detergents can be good for this. One example I know of and readily comes to mind is the use of 0.1% NP40 (Noniondet P40) in the stabilization of murine reverse transcriptase during purification (also helps toprevent precipitation), first described in: Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli., Roth MJ, Tanese N, Goff SP., J Biol Chem. 1985 Aug 5;260(16):9326-35. http://www.ncbi.nlm.nih.gov/pubmed/2410413 DDM may also be good, use at concentration much below CMC. Might be best to check out and screen several detergents, either by mini column purification or by DLS. Thanks, Debanu. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Vitali Stanevich Sent: Friday, October 12, 2012 9:42 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
The following paper (which can be found at www.wolfson.huji.ac.il/purification/PDF/Literature/Bondos2003.pdf Detection and prevention of protein aggregation before, during, and after purification. Sarah E. Bondos and Alicia Bicknell (2003) Analytical Biochemistry, 316, 223-231 contains a table of agents that may promote protein solubility on the 2nd page. Most are non-detergents which may be worth following up as well as Tween 80, Tween 20 and Nonidet P-40 at the recommended concentrations and their references. Dan
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
Vitali, Echoing what Dan said, I am not sure why you have chosen detergents first, as there are many other agents which stabilize proteins. Is the instability due to hydrophobic surfaces (e.g., made worse at higher salt) or not. Some non-detergent suggestions are: 1) diols like MPD (see work from Gil Prive's group) 2) non-detergent sulfobetaines (NDSBs), which is the head group in the zwittergent class of deterents. 3) Trimethylamine oxide, which is the head group in the amine oxide class of deterents (LDAO). 4) 200-500 mM L-Arginine 5) Also try 200-500 mM LiCl. The recommendations you received for detergents are very good ones, but remember that many of these detergents are quite dirty, as well as being chemically heterogeneous. Tween 80, Tween 20 and Nonidet P-40 are generally sold at industrial purity (i.e., they are delivered in tanker cars), so either clean them up yourself or buy purified samples (like the Pierce Surfact-Amps brand). Also, be aware that they may be hard to get rid of when you come to setting up crystals. Also look up Sam Gellman's work on detergent-assisted refolding for other stabilizing detergents. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Oct 12, 2012, at 12:42 PM, Vitali Stanevich wrote: Hi, Sorry for off-topic question. Does anyone have experience of the stabilisation of water-soluble proteins by detergents? Protein I'm working with is definitely water-soluble and has high yield, but, unfortunately, not very stable. Especially during concentration. So, we thought that adding some detergents may one of the ways to stabilise protein. So, did anyone do it before or may be know published examples? Any suggestions on the detergent type/concentration would be welcome. Thanks, Vitali
