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.

2.0.5 mM ATP,2 mM MgCl2,100 mM boric acid,pH 8.3 at 4℃,10 ml.

3.N-(1-pyrene)iodoacetamide (Molecular probes P-29,m.w.385)

4.50Ti tubes,small vial.

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

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

2.Add DTT (100 mM stock)to 5 mM.

3.Dialyze against 250 ml buffer 1 overnight.

4.Collect actin from dialysis tubing.Measure volume and bring the concentration of actin to 1.7 mg/ml with buffer 1.

5.Add 100 mM KCl and 2 mM MgCl2 to induce polymerization.Let sit at room temperature for 30 min.

6.Weigh about 1 mg pyrene iodoacetamide and place in a test tube.Dissolve in 100 µl DMSO.

7.Pipet buffer 2,with a volume equal to the volume of actin,into a small vial containing a stirring bar.Calculate the volume of pyrene iodoacetamide stock that contains 0.67 mg of the dye.While stirring,slowly add the calculated volume of pyrene iodoacetamide stock into the vial with a Pipetman.

8.Add the dye solution to actin and mix gently with a Pasteur pipet.

9.Wrap the container with aluminum foil and shake/rotate at room temperature for 16 hr.

Day 2

Materials

1.Buffer 1 as for day 1,4℃,2000 ml.

2.G-25-150 column,~30x1.5 cm.A G-150 column could be used for the simultaneous removal of protein contaminants.

3.50Ti tubes,volumetric conical tube.

Procedure

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

2.Soak the pellet for 1-2 hr in 0.5 ml of buffer 1.Dialyze overnight against 1500 ml of buffer 1.

3.Equilibrate G-25 column with Buffer 1.

Day 3

Materials

1.Ultrapure sucrose.

Procedure

1.Clarify dialyzed actin in a 50Ti rotor at 40,000 rpm,4℃ for 1 hr,or in a 42.2Ti rotor at 30,000 rpm,4℃ for 30 min.

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

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

4.Actin can be concentration by an additional cycle of polymerization-depolymerization.

5.Measure concentration and dye/protein molar ratio.Read the OD at 344 nm in an UV spectrophotometer.

D/P = {OD344 / 22,000} / {(mg/ml)/ 43,000},should be 0.9-1.0.

6.Calculate total mg of actin.Store as aliquots in liquid N2 after dissolving 2 mg sucrose per mg actin.

Reference

J.A.Cooper,S.B.Walker and T.D.Pollard (1983)Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization.J.Mus.Res.Cell Motil.4:253-262.

J.A.Cooper (1992)Actin filament assembly and organization in vitro.in The Cytoskeleton: A Practical Approach (K.L.Carraway and C.A.C.Carraway,eds),IRL Press,Oxford,pp.47-71.