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. |
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