klenow

Hi again,
I am trying to purify some protein for mass spectrometry and I have a problem. My protein has FLAG and HIS tags. When I check the supernatant of my cells (it is a secreted protein) by western-blot (after SDS-PAGE under denaturating conditions) I can see the main band of but, under certain conditions, I also see a higher molecular weight band. Another proteins from this family are reported to make covalent complexes with other proteins so I imaging that is what is happening with our protein of interest. This is extremelly interesting so I decided to pull down the protein using the HIS tag (to avoid having problems with heavy and light chains from anti-flag antibodies). The problem is that although I am able to pull-down the main band (single protein) the complex does not bind to the colum (well... only a tiny fraction of it can be detected after pull-down). I tried different buffers, salt, detergent, temperatures, etc, etc... and the result does not change.
I wonder if highly packed complexes bound by covalent bonds could, somehow, make the HIS tag unaccesible for the resin.... Does anyone experience something similar?
If I use denaturing conditions for the pull down (e.g. 8M Urea), do you think I would be able of pull the complex down? I used urea before for pull down a membrane protein but I do not know if covalent complex are urea-resistant...
Thank you for your help

Hi,
I was doing some pull-down and IP and it seems I treated all samples equally and instead of doing an elution step I directly added SDS-sample buffer containing B-mercaptoethanol to "realease" the complexes from the beads.
The problem is that some of the pull downs were HIS-tag proteins (I used HIS-tag dynabeads from invitrogen) but I did not add any imidazol. When I have realised of this, I had already added the SDS sample buffer and heated the samples (95C, 5min). It is possible to add the imidazole after this or SDS+B-mercapto+boiling it enough to release the complexes?
Thanks a lot,
Best.