Help with small protein with his-tag purification - (Aug/03/2011 )
i have a small protein with his-tag and tevsite. i have very nice expression but the problem is,that the protein no binding in to the Ni-NTA. the protein is in the soluble fraction. i trie many buffer and too witout imidazol but no work , i trie to in GdmCl 6M and no work, because for me is no importan if my amilin peptid 37 aa. is folding or no , but no work
try 8M urea
Have you checked if the his tag is in frame after cloning. Is the His tag N-terminal? If so, you can try cloning it C-terminal (or vice versa).
Dont use too much resin, i.e. histrap binds around 20-40 mg/ml. Try changing the Ni-NTA to Co-Talon or other metals with Talon. Whats your pH. I had similar problems with a protein when I had a 12xHis tag at the N-terminus (membrane protein). In this case my conclusion was that the tag was bound to something like a lipid when I purified in the presence of certain detergents which did not remove all of the tightly bound lipids. Your situation might be similar, i.e. a protein binds very tightly and occludes the tag.
Do you use reducing agent? NiNTA is not compatible with concentrations of DTT above 1mM and b-ME above 10mM.
Such a small protein/peptide is likely to be digested by proteases as well. Have you done western to confirm the expression and the protein's presence in the soluble fraction? I would consider changing the expression if you do not see it on the western probably by fusing it to MBP or so if you do E. coli over-expression.
I had the same problem. Mine was 10x his tagged to the furthest N-terminus of a membrane protein. I checked the expression level with western blot and binding experiments. I blamed that the N- 10X his were wrapped and therefore, not accessible to the resin. Is this reasoning valid? also wonder how to solve it?
N terminal tagging of membrane proteins is problematic. They often include an export tag which is cleaved, and the remaining C terminal fragment is lipidated to bind to the membrane. If your tag is on the N terminus, it likely is cleaved as well.
I would ensure your protein is being expressed first and your his tag is there by anti-His western blot