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Buffer in His Tag Isolation


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#1 Mafer

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Posted 12 March 2017 - 12:42 AM

Hi,

 

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?



#2 labtastic

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Posted 12 March 2017 - 05:56 PM

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.



#3 labtastic

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Posted 13 March 2017 - 08:42 AM

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.






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