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Buffer in His Tag Isolation - (Mar/12/2017 )



I would like to know if different buffer could be used between lysis and equilibration/binding to His Tag Isolation system?

I'm using HisPur cobalt resin and the protocol was in phosphate buffer, however, lysis for the protein I am working with (in other publications) was in citrate buffer.


Will they interfere?


Citrate buffer is generally used for low pH buffers (<6) but is ok-ish for pH 7.0 buffers. So check what pH you are using.


But even so, citrate is a good chelator of metals. It likely could chelate the cobalt (or nickle), which would make it a poor choice. Same reason why EDTA and EGTA are incompatible with his tag purifications.


So my vote is no, don't use citrate. Acceptable buffers for cobalt/nickel His tag purifications include phosphate, tris, hepes, pipes, mops...might be missing a few.


According to a manufacturer of nickel resins, up to 60 mM citrate is acceptable. But TALON resin is not the same thing and tends to be a bit more susceptible to chelation and incompatible reagents so maybe, maybe not.