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I was doing a protein prep and I made a mistake. After sonicating my cells, I was supposed to centrifuge and collect the supernatant (my protein is soluble and comes in the supernatant). I however, forgot to centrifuge and loaded the entire solution after sonication (debris and all) onto a Ni-NTA column.

Is there any way to dissolve the cell debris so that I may be able to recover the NTA beads?

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Follow the instructions that came with your column for cleaning. The resin will probably be fine, but you'll need to thoroughly clean the column. My GE columns recommend washes of:

  • 20% EtOH
  • 30% Isopropanol
  • 1M NaOH
  • 1M NaCL
  • SDS in 0.1M Acetic Acid
  • Stripping buffer (EDTA)

Wash between each with several column volumes of distilled water. You may not need to do all of them. Then just recharge your column with a 100 mM Ni solution (or metal of choice)

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You could try to resuspend everything and carefully decant, this would get rid of most of the cell material, as the beads will sediment much faster. Then wash the column with a ridiculous amount of acid or base.

But you should just discard everything and take new beads, they're not that expensive (maybe 10-20$/ml resin).

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  • $\begingroup$ You might also try washing with guanidinium. $\endgroup$ – canadianer Jul 11 '17 at 17:51

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