His Tag Protein Purification Problems

[IoDiNE]

Hey all,
i am currently working with a his-tagged protein of about 45KDa.

The protein has absolutely no solubility problem after overexpression from a pUC in 293 cells.

Column Binding Info:

This protein does not bind to Ni-NTA. With denaturation by 8M urea it binds to a certain extent in Ni-NTA.

For almost always the damn protein goes into the flow-through for any column you put it in.

I have tried DEAE, Ni-NTA, CM-Sepharose, and other columns with various pH ranges (8, 8.8, and pH 6.4 for CM Sepharose as well since the proteins theoretical pI is 10.02).

I confirmed this with western blot and anti-His antibodies in all the results.

any ideas???

thnx

[kmunson779]

I'd try tagging the opposite terminus to see if that end is more exposed.

Edited to add: Or -- have you tried binding in batch rather than on the column? It can give the protein more opportunity to bind. Or another thing -- you could try a cobalt resin rather than the nickel.