Imidazole cannot be used to elute the target protein in denaturing purification using 6M GuHCl, pH gradients are used instead. You can elute your target protein using 6M GuHCl buffer pH4.5. For example most commonly used denaturing sample

buffer is 100 mM NaH2PO4, 10 mM Tris–HCl, 6 M GuHCl, pH 8.0; denature washing

buffer is 100 mM NaH2PO4, 10 mM Tris–HCl, 6 M GuHCl, pH 6.3 and denature elution

buffer is 100 mM NaH2PO4, 10 mM Tris–HCl, 6 M GuHCl, pH 4.5.

Dear Paras

Guanidinium hydrochloride at 6 M is protein-denaturing. Precipitation of your protein of interest in its presence can be checked before smple application onto the affinity column you use.

Q1: What is it your objective? Purification of the protein in a denatured form?

Q2: What is it the pH value you use for the chromatography

Dear Joseph Kreit,

I want to purify protein in denaturing conditions, and after that check on western blot. 

I am not using columns, I am doing it in eppendorfs. 

I am doing chromatography