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/
-- 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 : cecile.brey...@ibs.fr
http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109
