1. 0.5 mM PMSF, 4oC. Need 10 ml per gram tissue. Prepare by diluting 100 mM PMSF stock in 95% ethanol.
2. Buffer A: 1 mM EGTA, 0.5 mM PMSF, 2 mM Tris-HCl, pH 9.0 at 4oC, 20 ml per gram tissue.
3. Buffer B: 0.1 mM EDTA, 0.5 mM DTT, 0.02% NaN3, 10 mM Tris-acetate, pH 7.6 at 4oC, 7000 ml.
4. PBS solution A, 2000 ml, 4oC.
5. 0.5 M acetic acid for titration.
6. Ultrapure (enzyme grade) ammonium sulfate.
7. DE-52 column, 2.5x20 cm.
8. Scalpels and dissection scissors.
9. Miscellaneous glassware, 2-liter graduate cylinders, 4-liter beakers.
10. 2 GSA rotors and Sorvalls.
11. Waring blender, prechilled.
1. Wash/rinse calf thymus in cold PBS. Remove fat and connective tissue. A typical preparation uses about 150 g (2-3 thymus, to fit into one GSA rotor). Extras can be stored frozen at -80oC. To use frozen gizzards, thaw for 1-2 hr in cold PBS at room temperature.
2. Homogenize in a Waring blender 3 times 10 sec bursts at high speed, in 10 volumes of 0.5 mM PMSF. Do this in the cold room.
3. Centrifuge in a GSA rotor at 12,000 rpm, 4oC for 60 min. Discard supernatant and floating lipids. Wipe lipids off the bottle.
4. Resuspend pellets in 10 volumes of buffer A at 4oC in a 4-liter beaker. Extract with gentle, constant stirring on ice for 1 hr. Check to make sure that pH is close to 9.
5. Titrate pH to 7.0 with 0.5 M acetic acid. Soak the electrode in high salt after use.
6. Centrifuge in a GSA rotor at 12,000 rpm at 4oC for 60 min.
7. Collect supernatant. Extract pellets as in steps 4-6, however extract for 40 min this time.
8. Pool both supernatants and measure volume.
9. Collect supernatnat and measure volume.
10. Bring ammonium sulfate to 15% saturation. Need 7.95 grams per 100 ml of volume in step 9. The crystals should be added slowly with constant stirring, while monitoring and maintaining pH at 7.0-7.2. Soak the electrode in high salt after this step.
11. Let stand on ice for 45 min after all ammonium sulfate has dissolved.
12. Centrifuge in a GSA rotor at 10,000 rpm, 4oC for 20 min.
13. Collect supernatant. Bring ammonium to 40% saturation as in step 10. Use 14.1 g per 100 ml as measured in step 9.
14. Let stand on ice for 45 min after all ammonium sulfate has dissolved.
15. Centrifuge in a GSA rotor at 10,000 rpm, 4oC for 20 min.
16. Resuspend pellets in 40 ml buffer B. Dialyze for 36 hr against 2 liters of buffer B, change buffer every 12 hr.
16. Equilibrate the DE-52 column with buffer B.
1. Buffer B, 2000 ml, degased.
2. Buffer P: 0.2 mM DTT, 0.02% NaN3, 50 mM (KH2PO4+K2HPO4), pH 7.0 at 4oC, 5 liters.
3. Whatman GFC filters, funnel, side-arm flask.
4. Hydroxylapatite column (BioRad DNA grade Bio-Gel HTP), 2.5x40 cm.
5. Gradient maker, fraction collector and UV monitor.
1. Collect solution from the dialysis bag. Centrifuge in a 50.2Ti rotor at 45,000 rpm, 4oC for 60 min. Discard milky portion of the supernatant.
2. Filter the supernatant through 2 layers of GFC filters under suction.
3. Clarify by centrifuging in a 50.2Ti rotor at 45,000 rpm, 4oC for 30 min.
4. Apply to the DE52 column equilibrated in buffer B.
5. Wash column with 100-200 ml buffer B or until OD drops to baseline.
6. Elute with 500 ml 0-450 mM NaCl gradient. Fill the mixing side of the gradient maker with 255 ml buffer B, the reservoir side with 245 ml buffer B with 6.5 g NaCl. Collect 3 ml fractions at 20-25 ml/hr. Set monitor full scale at OD 1.0, chart speed 1.5 cm/hr. Wash the column with 1 liter of 2 M NaCl in buffer B when finish.
7. Locate peak fractions. Run SDS-PAGE of various peaks. Pool alpha-actinin fractions towards the end of the gradient. Dialyze against 2 liters of buffer P for 24 hr. Change buffer at 12th hr.
7. Equilibrate the hydroxylapatite column with buffer P.
1. Buffer P, 2000 ml, degased.
2. Buffer B without EDTA, 5000 ml.
3. 1 M (KH2PO4+K2HPO4), pH 7.0, 100 ml.
4. Ultrapure (enzyme grade) ammonium sulfate.
5. Sepharose 6B-CL column, 2.5x90 cm.
6. Gradient maker, fraction collector and UV monitor.
1. Collect solution from the dialysis bag. Centrifuge in a 50.2Ti rotor at 45,000 rpm, 4oC for 1 hr.
2. Apply to the hydroxylapatite column equilibrated in buffer P. Wash with 50-100 ml buffer P or until OD drops to baseline.
3. Elute with 500 ml 50-300 mM phosphate gradient. Fill the mixing side of the gradient maker with 255 ml buffer P, reservoir side with 180 ml buffer P and 65 ml buffer 3. Elute at 20 ml/hr. Collect 3 ml fractions. Set monitor full scale at OD 0.5, chart speed 1.5 cm/hr. Wash the column with 1 liter of 500 mM phosphate in buffer P when finish.
4. Locate peak fractions and run SDS-PAGE. Pool alpha-actinin containing fractions (central peak). Measure volume.
5. Precipitate with 45% saturation ammonium sulfate (26 g per 100 ml).
The crystals should be added slowly with constant stirring, while monitoring and maintaining pH at 7.0-7.2. Soak the electrode in high salt after this step.
6. Let the solution stand on ice for 30-45 min after all ammonium sulfate has dissolved.
7. Collect precipitates by centrifugation in a SS34 rotor at 10,000 rpm, 4oC, for 20 min.
8. Resuspend pellets in 3 ml buffer B without EDTA. Dialyze against 2 liters of buffer B without EDTA for 24 hr. Change buffer at 12th hr.
9. Equilibrate Sepharose 6B-CL column with 1000 ml buffer B without EDTA.
1. Buffer B without EDTA, 2000 ml.
2. 2 mM PIPES, 0.02%NaN3, 1500 ml.
3. Ultrapure (enzyme grade) ammonium sulfate.
1. Collect solution from the dialysis bag. Centrifuge in a 50Ti rotor at 40,000 rpm, 4oC for 2 hr.
2. Apply to Sepharose 6B-CL column equilibrated in buffer B without EDTA.
3. Elute with maximum pressure. Collect 3 ml fractions. Set monitor full scale at OD 0.5, chart speed 1.5 cm/hr.
4. Run SDS-PAGE of peaks to locate alpha-actinin (central peak). Pool fractions and measure volume.
5. Precipitate alpha-actinin with 45% saturation ammonium sulfate (26 g per 100 ml).
6. Let the solution stand on ice for 30-45 min after all ammonium sulfate has dissolved.
7. Collect precipitates by centrafugation in a SS34 rotor at 10,000 rpm, 4oC, for 20 min.
8. Resuspend pellets in 400 µl of 2 mM PIPES with azide. Dialyze against 500 ml of this buffer. Change buffer twice over a period of 24 hr.
9. Clarify in a 50Ti rotor at 40,000 rpm, 4oC, for 45 min.
10. Store in liquid nitrogen as aliquots. Stable for months.