I eluted GST from a GST column using 10 mM GSH and removed excess GSH by dialysis. I am aware that GSH remains bound to GST. Does dialysis remove this as well? Will this GST bind the GST-resin again?
Yes that will work fine. We use GST beads to purify our recombinants then cleave the GST portion off and dialyse followed by rebinding to the beads to remove the GST leaving our protein in solution.
In our hands - No that will not work fine...
The GSH affinity is very high, and the off rate is supposedly extremely slow. Even long and multiple dialyses do not remove all GSH (at least, GST is not totally removed on glutathione column). Alternatives like ion exchange work better and quicker for us.
I went ahead and tried re-binding the tagged protein to the resin and it worked. I had to dialyze the protein multiple times to remove GSH.
I also had same problem of non-binding now i am trying with dialysis of the protein and rebinding....
- Badges
- Science method