I am having difficulty isolating a protein from its his tag. We have nailed down the correct vector and can cleave the tag off successfully. However, when we try to purify the cut protein from its tag, we are seeing our cut protein on the nickel agarose beads where the his tag is bound. We have tried introducing some imidazole and tween-20 to the buffer to try to reduce nonspecific interaction, in which case we saw some more of our cut protein in the sup, but at least half is still binding to the beads, which is unacceptable. I also am trying to make sure that any UNCUT protein doesn't separate into the sup as well.
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