Labeling Muscle Actin with 5-iodoacetamidofluorescein

Day 0 and 1

Materials

1.0.5 mM ATP,0.2 mM CaCl₂,2 mM Tris-HCl,pH 8.0 at 4℃,250 ml for day 0.

2.100 mM KCl,100 mM boric acid,pH 8.4 at 4℃,10 ml.

3.0.5 mM ATP,2 mM MgCl₂,100 mM KCl,2 mM DTT,2 mM PIPES,pH 7.0,1000 ml.

4.IAF (Molecular probes)

5.50Ti tubes,small vial.

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 (100 mM stock)to 5 mM.

3.Dialyze against 250 ml buffer 1 overnight.

4.Collect actin from dialysis tubing,add 100 mM KCl and 2 mM MgCl₂ to induce polymerization.Let sit on ice for 20 min.

5.Get 2.4 mg IAF in a test tube.Add 100-200 µl acetone and make a fine slurry by grinding particles against the tube with a pipet.Adjust the volume in step 6 if the amount actually weighed is higher.

6.Add IAF slurry very slowly by dipping a small amount at the tip of a Pasteur pipet into 2 ml of buffer 2 in a small vial under constant stirring.

7.Add 2 ml of the dye solution to actin and mix gently with a Pasteur pipte.

8.Let sit on ice for 2 hr.

9.Stop the reaction by adding DTT to 10 mM.

10.Dialyze against 1 liter buffer 3 overnight.

Day 2

Materials

1.Buffer 1 as for day 1,4℃,2000 ml.

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

3.50Ti tubes,volumetric conical tube.

4.Colloidin bag for dialysis.

Procedure

1.Equilibrate G-25 column with Buffer 1.

2.Pellet actin in a 50Ti rotor at 40,000 rpm,4℃,for 2 hr.

3.Rinse pellet briefly with buffer 1,soak and resuspend in 2 ml.

4.Dialyze against 500 ml buffer 1 at 4℃ for 4-5 hr in a colloidin bag.

5.Clarify dialyzed actin in a 50Ti rotor for 1 hr at 40,000 rpm,4℃.

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

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

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

9.Centrifuge in a 50Ti rotor for 2 hr at 40,000 rpm,15℃.

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

11.Dialyze against buffer 1 overnight.

Day 3 on

Materials

1.50Ti tubes.

Procedure

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

2.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},should be 0.5-0.9.

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

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