cycloheximide

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Thu Nov 11 22:21:21 EST 1999


In article <382B7DB6.A6577FA8 at mit.edu>, Irina <irka at mit.edu> wrote:
> Hi!
> Does anybody know a good protocol for inhibiting protein
> production in
> fibroblasts (or other TC cells) with cycloheximide?
> Also, how can I know (w/o using S35-methionine or other
> radioactively
> labeled a.a.'s) that the inhibition is happening.  Can you see it
> just
> by running a whole cell lysate on a gel and staining it with
> Coomassie
> blue?
> Thanks.
> Irina.

There's no special trick. Make your stock up to 10 mg/ml in water, PBS,
or whatever. Filter sterilize, if you want. Dilute into the cell
culture medium to a final concentration of 10-100 micrograms/ml. It
works almost as soon as you put it in.

You can pulse with radiocative met and count the TCA-precipitable cpms
to confirm that it's working. The "whole lysate on gel" is not
sensitive enough.

Nick



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