I'm working with a protein that forms different complexes. This is evident during a size exclusion run but lately, during the elution stage of a metal affinity chromatography run, the protein elutes at different stages. I usually don't run an imidazole gradient when eluting and usually the protein comes off all at once. Today, when I ran a gradient the protein comes off in two distinct places.
Any suggestions would be very welcome.
Best wishes
Harry
Hi Harry,
His-tagged oligomeric proteins can elute out in distinct places under an imidazole gradient. Different oligomeric states can potentially change the number of endogenous histidines exposed for nickel binding. Collecting all oligomeric fractions shouldn't pose a problem if you're planning to run size exclusion.
Collect the two peaks and analyze them with SE-UPLC, and SDS-PAGE, you may find that the protein either oligomerize or it is degraded or it is associated to other proteins.
Hi Harry
I have been working with a dimer protein which gave the same problem as you have stated. I was using N-terminal His tag for it. Adding another his tag at C-terminal solved this issue. The reasons may include different oligomeric state, partially buried His tag. Check both peaks on SDS PAGE, and use one or both depending upon their purity and your requirement.
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