Problems with elution of His Sumo tag protein from Probond resin

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Rahullin
Rahullin's picture
Problems with elution of His Sumo tag protein from Probond resin

Hi everyone...

This is my first post to Scientist solutions...

I am using Probond Sepharose(Invitrogen) for the purification of my His Sumo tag recombinant protein. Protein is bound on the beads but I cannot elute it out. I have used following ways.

Imidazole upto 500mM
Low pH upto 3.0 also High pH upto 10.0
High salt concentrations upto 2M NaCl
DTT upto 10mM
EDTA and EGTA upto 100mM

but still my protein of interest is on Probond Sepharose.

I see here following possibilities

I think it is very obvious that my protein itself has affinity for either IDA or sepharose itself or both.
At all these conditions I have tried , my protein should have eluted out if it is binding with only His tag or has own affinity for divalent cations.

I was wondering about how can I strip of IDA from sepharose or can someone suggest me some solution for this problem.
I highly appreciate it.