Hi :-) These subjects come up regularly! Basically, in brief we use a nearly magical mix of bper and yper in 3 to 1 ratio, supplemented with salt, bensonaze and lysozyme. In my fairly extensive experience this works for about 85 to 90 % of cytoplasmic proteins with no issues for affnity chromatography and (if no salt is added) for ion exchange primary capture. For the remaining 10% we try every other method under the sun. For gels - 15 minute biorad gels are very nice (have to have 300v power supply) and stain/destain in our hands is abou 5-15 minutes with a microwave. We destain in boiling water, with no other chemicals involved. Any coomassie based stin works well enough for this. Some ppl in our lab love the stain free gels but you have to remembr to count trp residues as it relies on trp chemistry to work. In short, this (and a number of other tricks) makes it possible to have a three step purification done in one 8 hr work day.
Artem On Mar 15, 2012 10:25 AM, "Thomas Edwards" <t.a.edwa...@leeds.ac.uk> 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 >
