By mechanical disruption you mean sonication only or have you tried the French press?
Assuming that you use sonication, and assuming that you follow a fairly standard protocol (e.g. something like 10sec pulse/20sec pause on ice for 3 minutes total), it may be heat not ultrasound that gets to it. Temperature in the sonicated sample may go pretty high under regular sonication protocol - we have the temperature probe and it easily goes over room temperature. I may certainly be wrong on both assumptions. And French press would suffer from the same heating problem - some pre-cool the whole thing in the cold room, but I don't know how much it helps. Cheers, Ed. On Thu, 2012-03-15 at 14:24 +0000, Thomas Edwards 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 -- "Hurry up before we all come back to our senses!" Julian, King of Lemurs
