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 <[email protected]> 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. > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [email protected] *******************************************
