View - ResearchGate

Affinity Precipitation of Proteins 45
5. Incubate at 30°C for 10 min. The precipitated protein–polymer complex is
centrifuged at 14,000 g for 5 min at room temperature (see Notes 13 and 14).
3.1.2. Washing Stage
1. Collect the supernatant and solubilize the protein–copolymer precipitate in 5 ml
of washing buffer containing 0.15 M NaCl.
2. Precipitate again by adding 1.5 ml 2 M NaCl and incubate at 30°C for 10 min. The
precipitate is collected by centrifugation at 14,000 g for 5 min at room temperature.
3.1.3. Recovery Stage
1. Dissociate the target protein from the protein–polymer complex by dissolving the
protein–polymer pellet in 5 ml of elution solution (50 mM EDTA buffer, pH 8 for
His-tag proteins, or 200 mM imidazole buffer, pH 7.4 for proteins with natural
metal-binding groups), while the mixture is kept on ice (see Note 15).
2. Precipitate the polymer by adding 1.5 ml 2 M NaCl and incubate at 30°C for 10
min leaving free target protein in the solution.
3. Collect the dissociated protein in the supernatant by centrifugation of the polymer
precipitate at 14,000 g for 5 min (see Note 16).
4. If required repeat the protein dissociation (see steps 1–3), to achieve complete
recovery of the bound protein (see Note 17).
5. Dialyze the recovered protein in 1lof10mMphosphate buffer, pH 7.4, or any
other buffer suitable for the protein to remove the metal ions leached out by EDTA
elution or imidazole buffer (see Note 18).
6. Determine the protein concentration by BCA method (36), using bovine serum
albumin as standard (see Note 19).
3.1.4. Recycling of the Metal Copolymer
1. Recover the metal–copolymer pellet and wash by dissolving and reprecipitating
again (see Subheading 3.1.3, steps 2 and 3).
2. Recycle the metal copolymer by dissolving the recovered polymer in 5 ml of
distilled water, pH 7, and use for further cycles of affinity precipitation of the
proteins (see Note 20).
3.1.5. Reloading with Metal
1. To increase the protein-binding capacity of the recycled metal copolymer to its
original capacity, reload the metal copolymers with fresh portions of metal ions.
Add 5 ml 0.01 M CuSO 4 or NiSO 4 to 10 ml of the recycled metal–copolymer
solutions, slowly while stirring. Stir and incubate metal-reloaded copolymers for
1 h at room temperature (see Note 20).
2. Wash the metal-reloaded copolymers by following the steps 2–5 as described in
Subheading 2.4.