His-tagged protein binding problem - Protein Binding (Apr/29/2005 )

I'm having a problem where I have managed to denature my HIS-tagged protein with Guanidine but when I purify using Ni or Co affinty beads only 1/3 of my protein binds to the resin, the rest still stays in the insoluble lystate. Any suggestions as to why most of it is not binding?

hi,

maybe the Ni-or Co- residues are saturated? You could try to use more.

By the way: 1/3 is not to bad!