We like using a BeadBeater (Biospec) to disrupt cells. It is actually
quite gentle, highly efficient, and very cost-effective. No potentially
harmful sound or aerosols. For gel staining we use a low-toxicity
ethanol-based stain-destain system. See our protocol wiki
<http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Electrophoresis+Protocols#Running_and_visualizing_SDS_PAGE_gels>at
capsicum.colgate.edu/chwiki.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [email protected]
On 3/15/2012 10:24 AM, 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
