LABELING MUSCLE ACTIN WITH CARBOXYFLUORESCEIN SUCCINIMIDYL ESTER

Day 0 and 1

Materials

1.0.5 mM ATP,0.2 mM CaCl2,1 mM PIPES,pH 6.8 at 4℃,250 ml for day 0.

2.0.5 mM ATP,0.2 mM CaCl2,70 mM KCl,150 mM PIPES,pH 7.0,1 ml.

3.16 mM ATP,0.2 mM CaCl2,5 mM DTT,5 mM lysine,10 mM glutamic acid,20 mM Tris-HCl,pH 8.0,10 ml.

4.3 mM CaCl2,1.2 M KI,50 mM Tris-HCl,pH 8.0,5 ml.

5.0.5 mM ATP,0.2 mM CaCl2,0.5 mM DTT,2 mM Tris-HCl,pH 8.0 at 4℃,1 liter.

6.CFSE (Molecular Probes,C-1311; not the diacetate),prepare a stock solution of 50 mg/ml in DMSO.

7.Small SS34 tubes and adaptors,50Ti tubes,small vial.

8.G-25-150 column,~30x1.5 cm.

Procedure (perform under reduced light,4℃ unless otherwise noted)

1.Resuspend 10 mg lyophilized actin in 2 ml buffer 1.Be careful not to make bubbles.

2.Add DTT to 0.5 mM.

3.Dialyze against 250 ml buffer 1 for 8 hrs or overnight.

4.Equilibrate G-25 column with buffer 5.

5.Collect actin from dialysis tubing and transfer to a small vial with a stir bar.Measure volume.Add KCl to 70 mM and CaCl2 to 1 mM.Incubate at room temperature for 30 min.

6.While vortexing,add 166 µl (8.31 mg)CFSE stock solution to 1 ml of buffer 2.

7.Mix dye solution immediately with actin solution,by gentle pipeting or stirring.Incubate at room temperature for 1 hr.

8.Centrifuge in a 50Ti rotor at 40,000 rpm,4℃ for 1 hr.

9.Resuspend pellet in 1.0 ml of buffer 3.Measure the total volume.

10.Add slowly an equal volume of buffer 4 while stirring.Stir gently on ice for 30 min.

11.Centrifuge in a SS34 rotor at 18,000 rpm,4℃ for 20 min.

12.Run supernatant through the G-25 column,collect 10 ｄｒｏｐ fractions.

13.Collect fluorescent fractions in the void volume,measure volume in a volumetric conical tube.

14.Polymerize actin by adding KCl to 100 mM and MgCl2 to 2 mM.Incubate for 30-60 min at room temperature.

15.Centrifuge in a 50Ti rotor for 1 hr at 40,000 rpm,15℃.

16.Soak pellet(s)in 0.6 ml buffer 5 for 1-2 hr,resuspend by gentle pipeting.

17.Dialyze against buffer 5 overnight.

Day 2 on

Materials

1.50Ti tubes.

2.Buffer 5 as for day 1,200 ml.

Procedure

1.Centrifuge in a 50Ti (1 hr,40,000 rpm)or 42.2Ti (30 min,25,000 rpm)at 4℃.

2.Polymerize actin by adding KCl to 100 mM and MgCl2 to 2 mM.Incubate for 30-60 min at room temperature.

3.Centrifuge in a 50Ti rotor for 1 hr at 40,000 rpm,15℃,or 42.2Ti rotor for 1/2 hr at 25,000 rpm.

4.Soak pellet(s)in 0.4 ml buffer 5 for 1-2 hr,resuspend by gentle pipeting.

5.Dialyze against buffer 5 overnight.

6.Centrifuge in a 50Ti (1 hr,40,000 rpm)or 42.2Ti (30 min,25,000 rpm)at 4℃.

7.Measure concentration and dye/protein molar ratio.Dilute 1:40 with the dialysis buffer and read the OD at 495 nm.

D/P = {OD495 x 41 / 60,000} / {(mg/ml)/ 43,000}

8.Dilute to 3-5 mg/ml with the dialysis buffer.Calculate total mg of actin.ｄｒｏｐ freeze in liquid N2 after dissolving 2 mg sucrose per mg actin.

9.Dialyze against 0.05 mM MgCl2,0.2 mM ATP,2 mM Tris-acetate,pH 6.95 overnight before microinjection.