Do you have a reducing agent in your solutions? I.e., maybe you are seeing disulfides?
JPK On Tue, Apr 12, 2011 at 1:27 PM, Michael Kenneth Fenwick <[email protected]> wrote: > Hi, > > I have a protein that shows high and low MW peaks on gel filtration (which > run at the same MW on SDS-PAGE). There is a slow equilibrium because > rerunning the individual peaks on gel filtration a couple days later shows > both peaks. The higher MW peak is ~2 orders of magnitude more dominant...the > lower MW peak is not yielding much. However, as nature would have it I've > only gotten the lower MW peak fractions to crystallize, and only when the > affinity tag is clipped (the higher MW species is resistant to clipping). I > would like to do something to shift the equilibrium towards the lower MW > species. So far, I've tried (without success or clues for success) changing > pH, increasing NaCl from 200 to 600mM, adding glycerol to 12%. Things that > I've seen in papers but have not yet tried are temperature jumps, other > salts, limited Gu/urea, Arg/glu, dioxane, limited SDS/triton. > > Any suggestions are greatly appreciated, thanks very much, > Mike -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [email protected] *******************************************
