More His-tag problems - (Apr/08/2010 )

Hey guys,

I have a major problem that I have been banging my head against a brick wall for (seemingly) months now. I have a protein which is His-tagged at the C-terminus. The protein induces very well, and I am attempting to purify it under denaturing conditions. However, no matter what I do the protein will not bind to the column (well, a very small amount binds, but literally almost zero).

The binding buffer that I'm using is simply 8M Urea, 100mM NaH2PO4 and 10mM Tris, pH8. I check the pH of the buffer everytime and yet it still seems to not bind. I have also tried it over a range of pHs and a range of salt concentrations but all to no avail. Do any of you have any ideas on what I can do to resolve this? (Also I have tried this with both N-terminal and C-terminal His tags, but again neither binds). At the minute I have only been doing this on a small scale using 50ul of Probond resin, and binding for up to 2 hours at RT on a rotator.

If anyone could help it would be most appreciated! I can't believe that the tag is unavailable in 8M urea!?

I do denaturing Ni++ pulldowns in 6M GuHCl. If the protein is there, I always get it.

I don't use a column though, I do it in bulk. After binding 2h-o/n, quick spin a wash 3x in urea to get rid of the GuHCl, this way it can be run on SDS-PAGE. Let me know if you'd like to see a protocol.

how many his tags are on your protein? probond prefers 6 or more.

are you sure the probond is still charged with nickel? have you regenerated?

the book on probond is to load and wash the protein in 8M urea, 0.5M nacl.

I would begin from the beginning. Are you sure that your construction contains the Histag?, have you done a western blot whith a monoclonal against the His-tag?, maybe it is not in frame if it is in the aminus-terminal or you have left a stop codon if it is in the C-terminal.

sumogirlie on Apr 10 2010, 08:52 AM said: