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
