Purification problems his-tag - I have tried everything (Apr/11/2010 )

Hello, this place has became my mentor. I suppose while completing the thesis I should get most of the credit for the awesome people in here Anyway... I'm doing my master thesis, still, and my supervisor is the worst possible ever. My ultimate goal is to create an antibody against c-terminal part of human protein. I cloned my insert from the human cDNA library. I put it into pET28 vector and expressed in E.coli. My protein formed inclusion bodies and was extracted by using BugBuster. It's soluble in 8M urea. I have proved that I have protein I want (correct size) with four different antibodies (don't ask me why I spent so much time with Western blotting to get four different antibodies to work to prove that I have the protein I want to make an antibody. I still don't know the answer, but it took me forever). I have tried to purify the protein by using protino ni-ted resin column. It worked out, somewhat, but the boss was not happy. While running the samples and doing Coomassie blue staining and silver staining I have some larger unwanted bands. I tried dialysis. My protein concentration somehow got really low. Then I ran my purified samples on the SDS-page and cut out the wanted protein bands out (gel extraction). While running the samples again on the gel I don't know how while cutting out 35-40kDa band I suddenly end up getting protein bands anywhere from 35-150kDa?!

I don't know what to do.
Boss says I'm in hurry.

I have tried to do gel extraction after BugBuster and as I had some unwanted bands, then column purification. The protein failed to bind to the column entirely.
I have done only column purification, I had some unwanted bands.
I have done column purification, gel extraction and somehow my cut out band size shows some totally different protein sizes on the gel.

I know E.coli bands shouldn't react with rabbits, in theory.
I don't know what the unwanted protein bands are after extraction.

I'm stuck and running out of time.
I should be finished in 2.5 months and yet I haven't even had the protein injected to the rabbit!
I need to get the protein. I have enough protein, but it's not pure enough and every single attempt to purify it has failed.

Maybe use a gradient elution and catch fractions off the nickle column? Your contaminating protein is retained by the nickle column, but it will likely come off earlier or later than your desired protein, under a correct elution profile. His-tag purification is a misnomer -- at best it's an enrichment...

When I was using his6 tag It didn't want to bind properly to the column. I would recommend his10 tag and I used to use those columns - http://www.gelifesciences.com/aptrix/upp01...cmpid=ppcaw1406 and I was collecting fractions.

Thank you for your replies... they helped a lot, even in the end I had a progress report and my boss sort of decided that the work I have done in the last six weeks all pointless and I was just wasting time, suddenly the extra bands didn't matter either (which were the big issue for weeks). What can I say... sometimes pleasing the boss is hard And I finally can move to the next step... this purification was driving me nuts