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Trouble With HIS affinity column (Ni+) binding

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#1 ithryn



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Posted 31 December 2009 - 09:53 AM

I've got a protein of interest (PDE4D) being expressed in bacteria, and I'm trying to purify it with a HIS affinity column (Sigma's HIS-Select) using a small-scale protocol.

I've verified the protein is being made via Western blot, and it's being translated in-frame from a vector with a His tag. But when I blot following purification by the column, the protein only lights up in the flowthrough faction, so it's apparently not binding through the column at all. (Otherwise it would at least show up in one of the two wash fractions, if not elution fractions.)

I've been extracting the protein using Novagen's Bugbuster solution, and using the standard recommended buffers with the column...

equilibration/wash - 50 mM sodium phosphate, 0.3 M sodium chloride pH 8
elution - equilibration + 250 mM imidazole

Is it commonplace to have to adjust the salt concentrations in buffers? What else can be tweaked?

I'm currently giving the 'denaturing conditions' protocol a go, with 8M urea buffers.

The ultimate aim is to purify said protein in order to compare Bradford assay results to my digital camera's program which quantifies bands on gels and blots based on relative intensity. Since Bradford and similar assays (BCA) are only indicative of total protein present, I've got to get it purified!

Edited by ithryn, 31 December 2009 - 09:58 AM.

#2 pDNA



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Posted 31 December 2009 - 01:34 PM

could be that the his-tag is "hidden" due to the conformation of the protein ...maybe denaturation will help. If that will not help you can try to tag it on the other terminus.


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