I purify my protein but in SDS-PAGE gel I have two proteins ( my protein and DNAk). I cant separate with Size exclusion column so I supposed both proteins are attached. So I want separated my protein of DNAk. How could do?. Thank you
Hi there,
You should try to add ATP in order to favor ADP/ATP exchange and therefore lower DnaK affinity for your protein.
As Dominique said, you can do a chaperone wash(buffer containing ATP) before elution of your protein. I normally do not get chaperone contamination after washing the column with chaperone buffer. In cases, when it is difficult to remove chaperones from my protein, I would go for ion exchange.
If you are purifying the protein using Ni-NTA, what you can do is add a wash step with a very high salt concentration (0.75-1M). It doesn't remove all the chaperone, but you can get rid of 90%.
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