I would like to purify my protein using His-tag affinity. Does the size of protein matter in his-tag purification? What is the maximum MW of a protein that can be purified using his-tag and nickel column or beads?
Thanks
Hi there,
size doesn't matter as long as his tag is accessible for affinity chromatography.
Agreed. Size is not a factor, per se, for affinity purification. I've used his-tags to purify protein ranging from 120 to 220kDa successfully. The challenge with larger proteins is that often they don't express well in recombinant systems. But there is no intrinsic problem with large proteins interacting with IMAC resins, provided your his tag is solvent accessible.
Thanks for the answers.
Daniel, I am just wondering if or not I can purify a protein about 1000 kDa. I am going to use Nickel magnetic beads. So, would it be possible to purify that much big protein?
Thanks.
Wow! The binding capacity of the resin might be limited here due to steric interference caused by such a large protein, but in principle this should work in terms of purification. Do you have any evidence that you can recombinantly express a protein of this size? Please post an update with your results if you are able to proceed- it will be interesting to see how this pans out.
yes, in principle i agree with Daniel , there is no limitation with the method, only main challenge is to express the protein and how to detect it. as far i know, the highest molecular weight protein which can be seen in gel is about 300 kDa.
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