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More His-tag problems


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#1 killmusic1979

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Posted 08 April 2010 - 04:18 AM

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!?

#2 sumogirlie

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Posted 09 April 2010 - 04:52 PM

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.

#3 mdfenko

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Posted 16 April 2010 - 09:11 AM

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.

Edited by mdfenko, 16 April 2010 - 09:12 AM.

talent does what it can
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#4 paramyosin

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Posted 26 May 2010 - 05:46 AM

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.
"Research without indebtedness is suspect, and somebody must always, somehow, be thanked." Umberto Eco

#5 AllenChiu

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Posted 31 May 2010 - 12:49 AM

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.

Hi,what did you do Ni pulldown for?to analyze Ubiquitination?




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