So what happened with the non-reducing gel? (If the DTT was fresh,
there should be no problem, but if not...)
JPK
On Tue, Apr 12, 2011 at 3:21 PM, Michael Kenneth Fenwick
<[log in to unmask]> wrote:
> Thanks for all your suggestions so far...as a quick reply to some:
>
>>You say the fractions are in equilibrium - how about keeping the oligomer fraction each time and adding it to the subsequent preparation?
> I did this once. The equilibrium is sort of a gift that keeps on giving, but the problem is the amount it's giving. In the end, this might be the only way to go, but it's very tempting to find a chemical solution.
>
>>Do you have a reducing agent in your solutions? I.e., maybe you are seeing disulfides?
> For native preps I used 1mM DTT throughout...for the SeMet prep I used 3mM DTT. To confirm it's not S-Ss, I'm about to run SDS-PAGE lacking reducing agent.
>
>>Changing buffer from Tris/Hepes into phosphate or citrate or acetate, or if higher pH borate?
> When I tried lower pH I used citrate. I might give the others a try.
>
>>Stay away from SDS/Triton because they will almost certainly kill your crystallization and it will be hard, very hard if not impossible to get rid
> of them.
> Thanks for the tip!
>
>>Another person off the bulletin board suggested differing how cell lysis is done
> I used sonication.
>
--
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Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
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