Purification of Capping Protein from Acanthamoeba
Dyche Mullins 11/98
1. Follow general protocol for amoeba prep using sucrose buffers. Elute protein from DEAE column with 0.0-1.0 M linear gradient of KCl in ATP- and calcium-containing buffer.
2. Collect and pool fractions containing capping protein. These are usually just before the actin. Determine position of capping protein by Elisa, falling-ball viscometry or actin elongation assays.
3. Concentrate 5-10 X by dialysis vs solid sucrose. Tie off bag and dialyze vs 50 mM KCl, 10 mM Imidazole (pH 7.5), 1 mM DTT, 0.01% NaN3 (capping protein buffer). Spin at 38,000 RPM for 1 hour in Ti-45 rotor at 4 C.
4. Run on 5 x 100 cm column of Sephacryl S-300 equilibrated in capping protein buffer. Assay for capping protein by immunoblot, ELISA, falling-ball viscometry, pyrene actin elongation or (on a calibrated column) take the fractions with a Stoke’s radius of 3.8 nm. Pool fractions containing capping protein.
5. Load S-300 pool onto a 1.5 x 5 cm column of hydroxylapatite (BioRad HT) equilibrated with capping protein buffer and elute with a linear gradient of 0.0-0.25 M K-phosphate. Pool based on capping activity and dizlyze into 10 mM Na-succinate (pH 5.5), 1 mM DTT, 0.01% NaN3.
6. Load HT pool onto charged phosphocellulose equilibrated with 10 mM Na-succinate (pH 5.5) and elute with a linear gradient from 0.0-0.3 M KCl. Pool based on activity and dialyze 2X vs capping protein buffer.
10X capping protein buffer: 1L
500 mM KCl 37.3 g
100 mM Imidazole (pH 7.5) 6.81 g
0.1% NaN3 1.0 g