His-tag protein expression problem - (Jan/10/2007 )
Im currently trying to purify two proteins of about 120kDa and 80kDa from mammalian cells. They are both the same His-tagged pcDNA plasmid. WB detects expression of my protein but not the His-tag, a postive control has been used so the anti-His Ab works. Nickel His-tag purification does not work. I have sequenced the plasmids again and the His-tag is in-frame at the N terminus, i was wondering whether anyone could give me any advice. I extract my protein using a triton lysis buffer
What elution buffer do u use ? Does it have imidazole and what conc. ?
pcDNA has myc. If ur plasmid has it, then try to detect using myc antibody.
Yeah the elution buffer has 500mM imidazole in it. The pcDNA im using doesn't have a myc tag
we use 250mM imidazole for elution. But u could try varying the concentrations of imidazole in ur binding or wash buffers.
We use 20mM or even 25 mM imidazole in our wash buffer (also the buffer used to extract).
Did u compare the flow thru of ur purification with the purified fraction using the antibodies. And did u compare it to a control extract.
these might suggest where the problem lies.
It sounds like your problem might be processing of the expressed protein.
The N-terminal his-tag might be cleaved off.
Try adding a tag to the C-terminal