If you suspect that your protein might form soluble aggregates in urea, you may try a stronger denaturant, such as guanidine huydrochloride or guanidine isocyanate

Hi there,

I have a silly question : did you make sure your protein is actually bearing the His-tag (using WB with anti-His antibody probing for instance)?

Is your protein is a membrane protein? If so then use the procedures for purification of membrane protein.

@ Pierre Béguin : Yes Sir, as till date results are driving me toward this conclusion. I am planning to use other denaturants. Any suggestion what concentration should I go with ?

@ Dominique Liger: Yes Dominique, I have tested my protein with Anti-His Ab. 

@ Vijayakumar Rajendran: Ok, I will see to it, Thanks

You could try saturated guanidine hydrochoride

6M Guanidine, 0.1M Tris-HCl pH 8 has worked well for me.  Be aware that you will need to heavily dilute samples in guanide in order to analyze them by SDS-PAGE. I also switch over to buffers with 8M urea for Ni-IMAC steps.

You can also add Tween -20, NP40 and lysozyme along with guanidium Hcl and still if you feel there are undesired results then maybe combine it with rapigest. and then try to separate it on column.  You should also check the concentration of Imidazole you are adding in the wash buffer!!!

Thanks Michael for the concentration. I was planning to go in gradient fashion as stated in Sambrook for IBs solubilisation. Initially washing and purifying at smaller concentration and then solubilising at 8M Guanidine concentration, do you think it will be too high ?

And yes I will dilute (only the sample volume to be loaded for SDS-PAGE). Should I dilute the whole sample before Ni-IMAC ?

and I plan to use Imidazole for Ni-IMAC steps, any comments ?

Thanks Nehil, will keep in mind. And I generally use lysozyme while initial lysis.

wash buffer range can vary from 50-100 mM IMD proceed further with an elution range of 250-500 mM  IMD. Cheers!!