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48 Kumar et al.
loosely bound metal ions. No pre-washing is needed with buffers containing
low amounts of imidazole, like 10–50 mM imidazole buffer ideally used in
traditional IMAC for pre-washing. Plain poly(NIPAM) shows almost negligible
non-specific interactions toward metal ions, and there is no entrapment of the
metal ions within the soluble polymer.
7. If precipitated pellets of copolymer take a long time for solubilization, incubate it
on ice and use glass rod to mechanically promote the dissolution of the polymer
pellet. Ensure that the NaCl concentrations entrapped inside the pellet is very
low, otherwise dilute by adding more water.
8. The capacity of the metal copolymer for binding the target protein needs to be
optimized by adding different amounts of the protein to the metal–copolymer
solution, and precipitation of the target protein above 90% is generally achieved.
9. Protein extracts preserved in azide or anti-proteases, such as benzamidine, should
be dialyzed to remove these compounds before applying to the metal copolymers,
as these can lead to poor precipitation/binding efficiency of the target protein.
10. Cell supernatants containing large amount of small peptides should also be
dialyzed to remove the peptides, which generally compete for the metal binding
and hence decrease the precipitation efficiency for the target protein (2).
11. Optimum precipitation of His-tagged proteins or proteins containing natural
metal-binding residues (especially histidines on the surface) with Cu(II)
copolymer can occur in the pH range of 6–7, whereas for Ni(II) copolymer, this
range can be slightly higher (pH 7–8) (2). Quantitative precipitation of the target
protein (above 90%) can be achieved in these pH ranges. On either side of this
optimum pH range, there can be decreases in the efficiency of precipitation of
the target molecules. Under acidic conditions below pH 6, the imidazole groups
in histidines are partially unprotonated (38) and hence show a low propensity
to coordinate metal ions. In alkaline conditions above pH 8, the decrease in the
selective binding of proteins is probably caused by the binding of other proteins
through increased competition for hydroxyl ions or coordination with partially
deprotonated -amino groups (10).
12. Cu(II) copolymers show higher capacity for protein precipitation than Ni(II)
copolymers, while the latter show slightly higher selectivity for the target protein
than Cu(II) copolymers (2).
13. Do not use refrigeration during centrifugation of the copolymer precipitates, as
it can solubilize the copolymer.
14. If the polymer precipitate is not sufficiently recovered during centrifugation,
make an empty run of the centrifuge (which can slightly increase the temperature
inside the centrifuge) before the precipitate is centrifuged.
15. The bound protein can be dissociated directly by dissolving the precipitate
of protein–metal–copolymer complex in elution buffer. His-tag proteins bind
strongly to the metal-loaded copolymers and are eluted only by using EDTA
buffer (2). The elution with imidazole buffer shows very low efficiency for
dissociating the His-tag proteins (2). On the other hand, imidazole buffer can