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Affinity Precipitation of Proteins 49
completely recover the proteins bound through natural metal-binding residues
(39,40).
16. To ensure that the polymer precipitate is efficiently precipitated and completely
recovered by centrifugation, warm the recovered supernatant in the presence of
0.4 M NaCl. That no visual turbidity changes occur in the supernatant means
the polymer is precipitated completely. If the supernatant turns cloudy, it means
that the polymer was not precipitated completely. In such cases, recover the
precipitate from the supernatant by slightly increasing both temperature and NaCl
concentration.
17. The metal affinity precipitation technique optimized in the present format using
the set of copolymers as discussed here can be essentially used for purifying
proteins that are relatively thermostable. However, it is possible to establish
copolymers with more hydrophobic side chains that can be utilized to carry out
precipitation at low temperatures as well.
18. The metal ions leached out with the recovered protein after EDTA or imidazole
elutions can be removed by dialysis. Determine the protein amounts or enzyme
activity of the recovered protein after the dialysis.
19. Protein measurements using BCA reagent show no interferences with high salt
concentrations or traces of polymers if present in the samples.
20. The metal poly(VI-co-NIPAM) copolymers recovered after the first use of affinity
precipitation of the protein can be reused for the precipitation of the same amount
of protein in the subsequent cycles, provided the copolymer is re-charged with
fresh portions of metal ions.
References
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