Switch ethanol in the stain. Also, there was talk a while ago about making your own "instant blue" stains--it works like bradford reagent, I think. Perhaps look back at previous postings?
JPK On Thu, Mar 15, 2012 at 9:24 AM, Thomas Edwards <[email protected]>wrote: > 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/ > -- No one should approach the temple of science with the soul of a money > changer. ~Thomas Browne > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: [email protected] *******************************************
