View - ResearchGate

44 Kumar et al.
5. Repeat the precipitation and dissolution of the copolymer as above (see step 4).
Measure the dry weight of the copolymer solution after drying the copolymer
solution at 80°C overnight.
6. Finally, dissolve the copolymer solution (see step 5) in water to give final 2%
(w/v, dry weight) copolymer solution.
2.4. Metal Loading to the Poly(VI-NIPAM) Copolymer
1. The Cu(II) and Ni(II) loading to the above copolymer solutions of poly(VI-
NIPAM) is carried out separately by adding an excess of copper or nickel sulfate
solutions as follows. Add 10 ml each of 0.1 M CuSO 4 or 0.1 M NiSO 4 solutions
to 20 mL of 2% poly(VI-co-NIPAM) solutions I and II, respectively, slowly while
stirring. Stir the metal ion-loaded copolymers for 1hatroom temperature (see
Notes 4 and 5).
2. Precipitate the metal-loaded copolymers by adding 2 M NaCl to a final concentration
of 0.4 M and heat at 40°C for 5 min during continuous mixing using a
glass rod (see Note 6).
3. Decant the supernatants and dissolve the precipitates of metal ion–copolymer
complex in 15 ml of water by stirring at 4°C till the copolymer is completely
solubilized (see Note 7).
4. Repeat the precipitation and dissolution step of the metal copolymer three times
as above (see Subheading 2.3, step 4) to completely wash out the unbound metal
ions (see Note 6). Determine the dry weight of the metal–copolymer solution after
drying the copolymer solutions at 80°C overnight.
5. Finally, the metal ion-loaded copolymers are dissolved in water to give a 2% (w/v,
dry weight) solution.
3. Methods
3.1. Purification of His-Tag Proteins or Proteins with Natural
Metal-Binding Groups
3.1.1. Binding Stage
1. Add protein extract (1–5 ml; depending upon the concentration of the target
protein) to 5 ml of the 2% metal ion–copolymer solution and make the total
volume up to 10 ml by adding the required volumes of distilled water (see Notes
8–10).
2. Keep the samples for a short period on ice (to prevent polymer precipitation) before
the pH is adjusted to 7 (for Cu(II) copolymers) and 7.5 (for Ni(II) copolymers)
(see Notes 11 and 12).
3. Incubate the polymer–protein mixture at 4°C for 30 min with constant mixing on
a rotating shaker.
4. Precipitate the protein–copolymer complex by adding 2.5 ml 2 M NaCl to give
final concentration of 0.4 M NaCl.