1. 0.5 mM PMSF, 4oC. Need 20 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 room temperature, bring to 37oC before use, 10 ml per gram tissue.
3. Buffer B: 20 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 0.02% NaN3, 20 mM Tris-acetate, pH 7.6 at 4oC, 4000 ml, or 1000 ml of 5x without DTT.
4. PBS solution A, 2000 ml, 4oC.
5. 0.5 M acetic acid for titration.
6. 3 M MgCl2 stock.
7. Ultrapure (enzyme grade) ammonium sulfate.
8. DE-52 column, 2.5x20 cm.
9. Scalpels and dissection scissors.
10. Miscellaneous glassware, 2-liter graduate cylinders, 4-liter beakers.
11. Two GSA rotors and Sorvalls.
12. Meat grinder, prechilled in a cold room and rinsed with 20 mM EDTA, pH 7.0 immediately before use.
13. High salt (e.g. 3 M KCl) for soaking electrode.
14. Polyron with prechilled large generator.
1. Wash/rinse chicken or turkey (preferred) gizzards in cold PBS. Remove connective tissue with a scalpel. A typical preparation uses about 300 g (to fit into 2 GSA rotors). Extras can be stored frozen at -80oC. To use frozen gizzards, thaw for 1-2 hr in cold PBS at room temperature until the gizzards become pliable.
2. Pass dissected gizzards twice through a meat grinder, using first the coarse then the fine mesh. Weigh the tissue.
3. Homogenize with Polytron 3 times 10 sec bursts, in 10 volumes of 0.5 mM PMSF. Do this in an ice bucket. Adjust the speed to obtain the maximal vortex.
4. Centrifuge in a GSA rotor at 10,000 rpm, 4oC for 10 min. Discard supernatant and floating lipids.
5. Resuspend pellets in 10 volumes of 0.5 mM PMSF. Homogenize in a Waring blender for 15 sec at low speed.
6. Centrifuge in a GSA rotor at 10,000 rpm, 4oC for 15 min. Warm up rotor after the spin.
7. Discard supernatnat. Wipe lipid off the centrifuge bottle. Resuspend pellets in 10 volumes of buffer A at 37oC in a 4-liter beaker. Extract with gentle, constant stirring in water bath at 37oC for 1 hr.
8. Centrifuge in a GSA rotor at 7,000 rpm at room temperature for 10 min.
9. Collect supernatnat and measure volume. Titrate supernatant to pH 7.0-7.2 with 0.5 M acetic acid.
10. Bring the concentration of MgCl2 to 10 mM. Use 3 M stock and add 1/300 of the volume measured in step 9. Stir at room temperature for 15 min.
11. Centrifuge in a GSA rotor at 7,000 rpm, room temperature for 10 min. Cool down the rotor to 4oC after this spin.
12. Carefully collect supernatant into a 4 liter beaker sitting on ice, avoid bubbles.
13. Bring ammonium sulfate to 40% saturation. Need 22.6 grams per 100 ml of volume in steps 9 and 10. 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.
14. Let stand on ice for 45 min after all ammonium sulfate has dissolved.
15. Centrifuge in a GSA rotor at 7,000 rpm, 4oC for 15 min.
16. Resuspend pellets in 20-30 ml buffer B. Dialyze for 36 hr against 1 liter of buffer B, change buffer every 12 hr.
17. Equilibrate the DE-52 column with buffer B.
1. Buffer B, 1500 ml, degased.
2. Buffer P: 0.2 mM DTT, 0.02% NaN3, 50 mM (KH2PO4+K2HPO4), pH 7.0 at 4oC, 5 liters.
3. Hydroxylapatite column (BioRad DNA grade Bio-Gel HTP), 2.5x40 cm. The size is important since HTP has a limited binding capacity.
4. 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 45 min.
2. Apply to the DE52 column equilibrated in buffer B.
3. Wash column with 100-200 ml buffer B or until OD drops to baseline.
4. Elute with 300 ml 20-370 mM NaCl gradient. Fill the mixing side of the gradient maker with 155 ml buffer B, the reservoir side with 145 ml buffer B with 3.9 g NaCl. Collect 3 ml fractions at 20-25 ml/hr. Set monitor full scale at OD 2.0, chart speed 1.5 cm/hr. Wash the column with 1 liter of 2 M NaCl in buffer B when finish.
5. Locate peak fractions. Run SDS-PAGE of various peaks. Pool vinculin fractions near the beginning of the gradient and follow vinculin preparation protocols.
6. 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, 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 35,000 rpm, 4oC for 2 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 4 ml fractions. Set monitor full scale at OD 2.0, 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 15 min.
8. Resuspend pellets in 4 ml buffer B. Dialyze against 2 liters of buffer B for 24 hr. Change buffer at 12th hr.
9. Equilibrate Sepharose 6B-CL column with 1000 ml buffer B.
1. Buffer B, 2000 ml.
2. 2 mM PIPES, 0.02% NaN3, 3000 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 45 min.
2. Apply to Sepharose 6B-CL column equilibrated in buffer B.
3. Elute with maximum pressure. Collect 3 ml fractions. Set monitor full scale at OD 2.0, chart speed 1.5 cm/hr.
4. Run SDS-PAGE of peaks to locate alpha-actinin (central peak). Pool fractions and measure volume.
5. Concentrate with CentriPrep or with ammonium sulfate precipitation: precipitate alpha-actinin 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), let the solution stand on ice for 30-45 min after all ammonium sulfate has dissolved, and collect precipitates by centrafugation in a SS34 rotor at 10,000 rpm, 4oC, for 15 min.
6. Resuspend pellets in 3 ml of 2 mM PIPES with azide. Add DTT to 5 mM. Dialyze against 1000 ml of 2 mM PIPES. Change buffer twice over a period of 24 hr.
7. Clarify in a 50Ti rotor at 40,000 rpm, 4oC, for 45 min.
8. Store in liquid nitrogen as aliquots. Stable for months.
1. Buffer C: 20 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA, 0.02% NaN3, 20 mM NaAc, pH 5.0 at 4oC, 5000 ml, or 1000 ml 5x without DTT.
2. 5 mg/ml PMSF, 5 mg/ml leupeptin in 95% ethanol, 0.3-0.5 ml.
3. CM-52 column, 1.5x40 cm.
1. Carry through alpha-actinin preparation until day 3-4 step 5. Measure the volume of vinculin-containing fractions.
2. Add PMSF and leupeptin very slowly, while stirring, to a final concentration of 0.1 mg/ml.
3. Dialyze against 2 liters of buffer C for 24 hr. Change buffer at 12th hr. The solution turns cloudy transiently as it passes across the isoelectric point of vinculin.
4. Equilibrate CM-52 column with buffer C.
1. Buffer C, 1000 ml.
2. Ultrapure (enzyme grade) ammonium sulfate.
3. 2 mM PIPES, 0.02% NaN3 1500 ml.
4. High salt (e.g. 3 M KCl) for soaking electrode.
1. Collect solution from the dialysis bag. Centrifuge in a 50Ti or 50.2Ti rotor at 35,000 rpm, 4oC for 2 hr.
2. Apply to CM-52 column equilibrated with buffer C.
3. Wash with 50-100 ml buffer C or until OD drops to baseline.
4. Elute with 500 ml 200-500 mM NaCl gradient in buffer C. Fill the mixing side of the gradient maker with 255 ml of buffer C with 2.9 g NaCl, the reservoir side with 245 ml buffer C with 7.2 g NaCl. Elute at 10 ml/hr. Collect 4 ml fractions. Set monitor full scale at OD 0.5, chart speed 1.5 cm/hr. Wash the column with 2 M NaCl in buffer C when finish.
5. Locate peaks and run SDS-PAGE. Pool vinculin fractions (central peak).
6. Carefully bring pH close to 7.0 with 0.1 N KOH. Soak electrode in high salt after this step.
7. Concentrate with CentriPrep or concentrate with the ammonium precipitation method: measure volume and 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). Let the solution stand on ice for 30-45 min after all ammonium sulfate has dissolved, collect precipitates by centrafugation in a SS34 rotor at 10,000 rpm, 4oC, for 15 min. Resuspend pellets in 300-700 µl of 2 mM PIPES with azide.
8. Add DTT to 5 mM. Dialyze against 500 ml of 2 mM PIPES. Change buffer twice over a period of 24 hr.
9. Clarify in a 50Ti rotor at 40,000 rpm, 4oC for 45 min, or in a 42.2Ti rotor at 25,000 rpm, 4oC for 20 min. Concentrate with Centricon if necessary.
10. Store in liquid nitrogen as aliquots. Stable for months. Also stable at 4oC for a couple of weeks.