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 MgCland 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.