Labeling Gizzard Myosin with Tetramethylrhodamine Iodoacetamide (Primarily 17kDa Light Chain)

Day 1

Materials

1.50 mM KCl,0.2 mM EGTA,2 mM ATP,2 mM MgCl₂,10 mM HEPES,pH 7.0.Need 500 ml.

Procedure

1.Thaw appropriate volume of frozen gizzard myosin to obtain 10-20 mg.

2.Centrifuge in a SS34 rotor at 9,000 rpm,4℃ for 20 min.

3.Resuspend pellet in a small volume of buffer 1 to obtain 15-20 mg/ml concentration.Dialyze against 500 ml of buffer 1 for >=2 hours.

4.Measure volume.Dilute with dialysis buffer to obtain a myosin solution of 12-15 mg/ml.

5.Get an aliquot of IATR,dissolve in DMF or DMSO at 7 mg/ml.The amount needed for reaction is 0.12 mg IATR per 10 mg of myosin.Add the calculated volume very slowly to myosin.Mix gently.

6.React on ice for 6 hours.

Day 2 on

Materials

1.50 mM KCl,0.2 mM EGTA,1 mM DTT,10 mM HEPES,pH 7.0.Need 2 liters.

2.Buffer 1 with 10 mM MgCl₂,10 ml.

3.Buffer 1 with 2 mM MgCl₂,2 mM ATP,10 ml.

4.50 mM KCl,0.2 mM MgCl₂,0.2 mM ATP,0.1 mM DTT,20 mM Tris-acetate,pH 7.0.Need 250 ml.

Procedure

1.Add DTT to 10 mM.Centrifuge in a 50Ti rotor at 30,000 rpm,4℃ for 30 min.

2.Dialyze supernatant against buffer 1 at 4℃ for >=3 hours.

3.Measure the volume and bring MgCl₂ to 10 mM.The solution should turn turbid.Allow solution to sit on ice for 30 min.

4.Centrifuge in a SS34 rotor at 9,000 rpm,4℃ for 15 min.

5.Resuspend pellets in 3-5 ml of buffer 2,centrifuge as in step 4.

6.Resuspend pellets in a small volume of buffer 3.

7.Dialyze overnight against buffer 4.

8.Centrifuge in a 50Ti rotor at 35,000 rpm,4℃,for 60 min.The supernatant is good for microinjection for 2-3 days.Most of the fluorophores are located at 17 kD mlC.