LABELING MUSCLE ACTIN WITH IATR

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 and 1000 ml for day 1.

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

3.IATR (tetramethylrhodamine iodoacetamide; Molecular Probes).

4.50Ti 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 >=1 mg IATR in a test tube.Add 100-200 µl acetone and make a fine slurry by grinding particles against the tube with a pipet.

6.Add IATR slurry very slowly by dipping a small amount at the tip of a Pasteur pipet into 3 ml of buffer 2 in a small vial under constant stirring.

7.Clarify in a 50Ti rotor at 40,000 rpm for 10 min,4℃.

8.Collect clarified dye solution.Dilute 5 µl into 1 ml distilled water and measure OD at 555 nm.The OD should be in the range of 0.05 to 0.10.

9.(optional)Calculate the volume (ml)of IATR required for reaction as 0.15 / OD or use an volume equal to that of actin.Mix gently with actin with a Pasteur pipet.

10.Let sit on ice for 2 hr.

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

12.Dialyze against 1 liter buffer 1 overnight.

Day 2

Materials

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

2.Buffer 1 as for day 1,4℃,1000 ml.

3.50Ti tubes,volumetric conical tube.

Procedure

1.Equilibrate G-25 column with Buffer 1.

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

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

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

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

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

7.Soak pellet(s)in 0.4 ml Buffer 1 for 1-2 hr,resuspend by gentle pipeting.Sometimes there is a dark center on the surface of the pipet,which can come off as an aggregate when handled gently.Discard the aggregate by careful pipeting.

8.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 555 nm.

D/P = {OD₅₅₅ x 41 / 60,000} / {(mg/ml)/ 43,000},should be 0.5-1.1.

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.