Purification of α-actinin from Acanthamoeba
Dyche Mullins 11/98
1. Follow general protocol for amoeba prep using sucrose buffers. Elute protein from DEAE column with 0.0-0.6 M linear gradient of KCl in ATP- and calcium-containing buffer.
2. Collect and pool gel-ed fractions containing actin and a-actinin. These usually have conductivity between 7-11 mMHO or KCl concentration around 0.3 M. Conscientious method: Determine positions of actin and a-actinin by Elisa. Dirtball method: Allow fractions to sit for 1-2 days in the cold room and pool obviously gel-ed fractions
3. Add 2 mM MgCl2 and warm fractions to room temperature to polymerize actin. Incubate 1 hour at 24 C with stirring.
4. Spin down actin for 90 minutes at 4 C at 38,000 RPM in Ti-45 rotor. Keep pellets for actin prep. Take sups for a-actinin prep.
5. Add solid ammonium sulfate to 1 M and incubate with stirring at 4 C for 4-6 hours. Spin out precipitate at 3,000 RPM for 30 minutes. Retain supernatant. Keep pellet just in case.
6. Add additional ammonium sulfate to supernatant to a total concentration of 2.3 M and incubate with stirring overnight. Spin out precipitate at 3,000 RPM for 30 minutes. Keep supernatant just in case.
7. Resuspend pellet in 0.8 M KCl, 10 mM Imidazole (pH 7.5), 1 mM CaCl2, 1 mM DTT, 0.01% NaN3. Dialyze vs. this buffer o/n with 1-2 changes.
8. Add solid KI to 0.6 M and homogenize the sample in a Dounce homogenizer with a tight-fitting plunger. Spin at 38,000 RPM in Ti-45 rotor for 30 minutes at 4 C.
9. Run on 4×100 cm Sephacryl S-300 equilibrated with 0.6 M KI, 10 mM Imidazole (pH 7.5), 1 mM DTT, 0.01% NaN3. Assay fractions for α-actinin by SDS-PAGE, ELISA, or falling-ball viscometry and pool. Alternately, on a calibrated column take the fractions corresponding to a Stoke’s radius of 8.5 nm.
10. Dialyze 3-4X vs. 1L of 10 mM Imidazole, 1 mM DTT, 0.01% NaN3. Load onto 2.5 x 7 cm column of hydroxlapatite (BioRad HT) equilibrated with the same buffer. Elute with a linear gradient of 0-100 mM K-phosphate in a total of 300 mL column buffer. Assay fractions by SDS-PAGE or falling ball viscometry. α-actinin usually comes out in the first peak. Dialyze vs 1 L 10 mM Imidazole (pH 7.5), 1 mM DTT, 0.01% NaN3. After this step the α-actinin is clean enough for most purposes.
11. To remove the final minor contaminants, run the α-actinin on a 2.5 x 7 cm column of DEAE and elute with a linear gradient of 0-400 mM KCl in a total volume of 300 ml column buffer.