Any idea of the affinity of the complex? If weak enough, you could just dialyse, dialyse, dialyse... Or, you could bind your protein to a column, and wash with a slow-flow of appropriate buffer. Or you could also try to compete it out with adenosine, for example, which presumably has a lower affinity, but at high concentrations would still compete it out, and then dialyse or rinse while bound to a column.
Jacob On Fri, May 20, 2011 at 10:18 PM, jlliu liu <[email protected]> wrote: > Hi All, > I am working with a methyltransferse protein and its later determined > crystal structure indicates its substrate SAM was bound during protein > expression or purification since no SAM was added during protein > crystallization. Now I want to obtain the apo form protein for further > characterization. Here are the strategies I can think of: > 1. Mutate residues that are possibly critical for SAM binding. The > shortcoming of this strategy is the protein is modified, also mutating one > residue may or may not be able to get rid of SAM binding. > 2. partially denature protein with denaturing reagent such as guanidine. > This strategy is hoping the partial denaturing would loose SAM binding, then > dialysis out SAM, reconcentrate protein and re-run sizing column to get rid > of aggregated protein. This strategy may only work with well-behaved > protein. The harsh denaturing reagent treatment could lead to total protein > denaturing. > I am asking more experienced biochemists if there are other better strategis > for obtaining apo protein that I am not aware of. Thanks very much for > sharing your knowledge. > Eric -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [email protected] *******************************************
