Hey, that was my post! NB this cannot be used for native gels, I don't think.
Jacob On Thu, Mar 15, 2012 at 9:47 AM, Cécile Breyton <[email protected]>wrote: > Hi Ed, > > A few years back there has been a thread on copper staining that leads to > "negative staining", but works well, is very fast and simple. > > - Rinse gel 10-30s in H2O > - incubate in 300 mM CuCl2. The gel stains in 3-5 min: background becomes > opaque, the protein bands remain clear. Sensibility is as good (slightly > better) than Coomassie. > > The gel can be stored in H2O (but proteins diffuse after a while), > destained for other staining (Coomassie, silver...), by incubation in 50 mM > EDTA 5-10 min. > > CuCl2 and EDTA solutions can be re-used. > > Discard Cu in appropriate waste. > > From Lee et al, 1987, Anal. Biochem 166, 308-312. > > Cécile > > > Le 15/03/12 15:24, Thomas Edwards a écrit : > > Dear BB, >> >> Apologies for being mildly off topic. >> Maybe. >> >> >> 1. We are trying to express (in E. coli) a protein which appears to be >> quite sensitive to mechanical disruption. We have ordered some B-PER >> (Pierce - "B-PER Bacterial Protein Extraction Reagents are designed to >> extract soluble protein from bacterial cells without harsh chemicals or >> mechanical procedures like sonication"), but would like to try a variety of >> similar things if possible. Any advice from the community out there? >> Anybody know what goes into B-PER or similar things (I know there's some >> Dnase and lysosyme in there – but which detergents are compatible with Ni, >> GST, how much do you need etc)?? >> 2. Staining SDS gels. There are various concerns from lab members about >> safety re methanol in stains, microwaving stains etc etc. "Instant Blue" >> claims to have none of these problems. Quote: "Protein gel staining takes >> around 15 minutes without the need to wash, fix, microwave or destain". But >> again, I'd like to try things to see if they work for us (before spending >> cash - yes, I am spending averse…!). Anybody any suggestions for quick, >> non-fix, non-methanol, non-microwave, non–destain protein gel stains? Have >> tried home made colloidal coomassie but our protocol still requires fixes >> and washes that made it not really worth while. >> >> Happy to collate thoughts on replies offline and post summary. >> >> Many thanks >> Ed >> >> T.A.Edwards Ph.D. >> Deputy Director Astbury Centre for Structural Molecular Biology >> Lecturer in Biochemistry >> Garstang 8.53d >> University of Leeds, Leeds, LS2 9JT >> Telephone: 0113 343 3031 >> http://www.bmb.leeds.ac.uk/**staff/tae/<http://www.bmb.leeds.ac.uk/staff/tae/> >> -- No one should approach the temple of science with the soul of a money >> changer. ~Thomas Browne >> >> > -- > Cécile Breyton > Institut de Biologie Structurale > UMR 5075 CNRS/CEA/UJF > 41, rue Jules Horowitz > 38027 Grenoble cedex 1 France > --- > Tel: +33 (0)4 38 78 30 37 > Fax: +33 (0)4 38 78 54 94 > Courriel : [email protected] > http://www.ibs.fr/groups/**membrane-and-pathogens-group/** > ssimpa/article/ssimpas-1109<http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109> > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: [email protected] *******************************************
