I'm purifying a protein (~27kDa) using Ni-NTA (Qiagen) and have hit a problem: When using the spin-columns (Qiagen), the results are textbook and the gels come out beautifully. When scaling up to the Ni-NTA agarose (not the superflow), I can't seem to find my protein anywhere (even after boiling the beads) which leads me to believe that maybe the protein isn't binding to the resin at all. Any idea why the kit would work (which still requires an exposed 6xHis Tag) while the column/batch procedure wouldn't using the same buffers?
Any help would be appreciated.
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