Hi, we also stain gels in microwave: Comassie with 5% acetic acid to stain and just water for destaining. We keep the microwave under the fan to avoid breathing acetic acid.
For E. coli cell disruption we are quite happy with Emulsiflex C3 by Avestin. In many cases proteins that undergo proteolysis, precipitation or instability during purification (after sonication) behave very well using Emulsiflex. It requires some maintenance but it solves many problems. Claudia 2012/3/15 Thomas Edwards <[email protected]> > 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 > -- Claudia Binda University of Pavia Dept. Biology and Biotechnology via Ferrata 1, 27100 Pavia - Italy Phone: +39-0382-985535 Fax: +39-0382-528496 E-mail: [email protected] Web: http://www.unipv.it/biocry
