1. 1 mM EDTA, 0.1 mM ATP, 0.1 mM PMSF (from stock of 100 mM in 95% alcohol, highly toxic), 0.5 mM DTT, 10 mM imidazole-HCl, pH 7.0, 4oC. Need 3 ml per gram wet tissue.
2. 100 mM glycine, 100 mM NaCl, 1 mM DTT, 10 mM Tris-HCl, pH 7.8, 4oC. Need 15 liters. Make 10x, dilute and titrate pH.
3. PBS solution A, 1000-2000 ml, 4oC.
4. Poly-L-proline affinity column, 2.5x15 cm. Conjugate poly-L-proline (m.w. 10,000, Sigma P-2129) to CNBr-Sepharose. Pre-equilibrate with buffer 2 at 4oC. The column can be used repeatedly for many preparations. However, it should be cycled batchwise through acidic and basic buffers when there is a noticable reduction in flow rate. See steps 12-14 in the procedure for conjugating proteins to sepharose.
5. Waring blender, prechilled.
6. Fraction collector and UV monitor.
7. 3 M urea in buffer 2, 400 ml.
8. 8 M urea in buffer 2, 400 ml.
9. Type 35 rotor and bottles.
1. Rinse out blood with PBS and trim brain in the cold room. Remove blood vessels, connective tissue, and white lipids. Trimmed tissue can be stored at -80oC. When using frozen tissue, let it thaw in cold PBS over 1-2 hr at room temperature. Weigh the cleaned tissue. Start with 500-700 g.
2. Homogenize in 3 volumes of buffer 1, using several short bursts (~5 sec) in a Waring blender at a medium to high speed in the cold room.
3. Centrifuge in a GSA rotor at 11,000 rpm, 4oC for 1 hr.
4. Collect supernatant and centrifuge in a Type 35 rotor at 33,000 rpm, 4oC for 3 hr.
5. Load supernatant into the poly-L-proline column overnight. The maximum operating pressure for Sepharose 4B is 80 cm H2O.
6. Wash the column with 1200 ml buffer 2.
7. Wash with 3 M urea in buffer 2.
8. Elute with 8 M urea in buffer 2. Set sensitivity of UV monitor at 0.5. The column can be regenrated after extensive wash with buffer 2.
9. Collect peak fractions. Dialyze against 4 liters of buffer 2 at 4oC, change buffer 2 times over a 36 hr period.
10. Concentrate profilin in a colloidin bag with m.w. 10,000 cutoff, by either vacuum dialysis or aquacide II. Should yield ~0.5 mg profilin from 100 g wet tissue. Store in liquid nitrogen.
D.A.Kaiser, P.J.Goldschmidt-Clermont, B.A.Levine and T.D.Pollard (1989) Characterization of renatured profilin purified by urea elution from poly-L-proline agarose columns. Cell Motil. Cytoskeleton 14:251-262.
1. See profilin preparation, items 1, 3, 4.
2. Buffer 2 as for profilin preparation, but without DTT.
3. 30% DMSO in buffer 2 without DTT, 400 ml.
4. Centriprep-30.
1. Load the poly-L-proline column as for the preparation of profilin.
2. Wash the column with 1200 ml buffer 2 without DTT.
3. Elute the column with 30% DMSO in buffer 2. The column cannot be regenerated after use with DMSO.
4. Collect peak fractions. Dialyze against 4 liters of buffer 2 at 4oC, change buffer 2 times over a 36 hr period. DTT is added for the second and third buffer .
5. Concentrate with Centriprep-30. Free profilin should go through the filter. Store in liquid nitrogen.
M.Rozycki, C.E.Schutt and U.Lindberg (1991) Affinity chromatography-based purification of profilin:actin. Methods Enzymol. 196:100-118.