His tagged protein won't elute from nickel resin - (Sep/08/2006 )

So I have expressed my His10-tagged protein in coli in inclusion bodies, solubilised protein in 8M urea.

I passed the lysate over Ni-CAM resin (sigma) in a small-scale batch prep, it wouldn't elute even in pH4.5 (still 8M urea). But then I left the resin in the coldroom overnight in the elution buffer pH4.5 and the pure protein was in the supernatant next morning!

I tried to upscale this using a column and some more material, but now it just refuses to come off the column. I even tried stripping the nickel from the column using 3 washes of 100mM EDTA, but only a minor portion of the protein came away. (I didn't notice the column change colour at the time but it now appears as a dull grey when I compare it to the fresh resin which is blue). Apart from boiling the resin in Laemli buffer, nothing get much of my protein off. I'm beggining to think that it may have precipitated onto the resin in the coldroom (perhaps the urea conc dropped due to urea precip.?) but if so it should be re-solubilized in 8M urea buffers I am using at room temp.

Any ideas? Can I heat this resin without destroying it? (it is 6% beaded agarose, I wonder if I could elute at 50 degrees..)

We use 250mM imidazole when eluting thru nickel column.

The protein is binding directly to the resin...........if you are using any other method besides immidazole, pH or EDTA to elute it off it would mean that you are not purifying the protein based on the his tag principle............

I'm currently experiencing that difficulty................