Tom, What residues are being phosphorylated - S/T or Y?
You can take the phosphates off after/during purification but it is in fact easier to take them off while they're being put on (i.e. by expressing the kinase in E. coli that is already expressing a phosphatase). This tends to work much better - post-expression treatment does not usually remove all the phosphates (at least from a native protein - with unfolding you can get rid of them all, but that's not very elegant). Artem On Thu, Dec 23, 2010 at 11:31 AM, Brett, Thomas <[email protected]>wrote: > Hello: > I am making a kinase in e.coli that seems to autophosphorylate itself a > bit, which makes it difficult to get a pure homogeneous sample for > crystallization. I would like to use a phosphatase to remove the abberant > phosphorylation. I have seen lambda phosphatase used for this type of thing > in the past, but buying it is pricey. Does anyone have a e coli construct > for this or some other phosphatase or perhaps another suggested solution to > this dilema? thanks in advance. > -Tom >
