If your proteins precipitate in reversed phase solvents, then there may not
be much hope for keeping them afloat in aqueous buffers without denaturants.
You might try reduction/alkylation in a small volume of guanidine (or urea),
then diluting to a denaturant concentration at which your protease will
digest the target. (You may need to quench the alkylation reagent with some
scavenger if it interferes with proteolysis.) You'd be surprised how much
urea or guanidine some proteases can tolerate, and it may even speed up some
of your digestions.
Mike Klein
Amgen, Inc.
> ----------
> From: novick@immunex.com[SMTP:novick@immunex.com]
> Sent: Thursday, February 04, 1999 4:24 PM
> To: Recipients of ABRF List
> Subject: desalting
>
>
>
>
>
>
> Hi All,
> I am reducing and alkylating proteins in a guanidine solution and
> then
> buffer exchanging using the BioRad P6 centrifuge columns. Our group
> is
> looking for a faster, easier way to clean the sample (we can have
> many
> samples to desalt at a time). RP desalting doesn't work (protein
> precipitates and the protocol is WAY too long). I'm interested in
> some
> kind of desalting pipet that will make this a none-issue for the QC
> and Formulations folk. Are there any suggestions? The final solution
> should be in a neutral, enzyme-friendly, somewhat buffered solution.
> Thanks in advance for your help,
> Shawn Novick
>
>