LABELING GIZZARD MYOSIN WITH IATR (MHC AND 17KD MLC)

Day 1-2

Materials

1.0.5 M KCl,10 mM HEPES,pH 7.5,4℃,250 ml.

2.0.5 M KCl,50 mM HEPES,pH 8.0,4℃,250 ml.

3.Bio-Beads SM-2 in a 0.7x15 column.

4.20 mM KCl,20 mM PIPES,pH 7.0,4℃,250 ml.

5.IATR (tetramethylrhodamine iodoacetamide; Molecular Probes).

Procedure

1.Thaw appropriate volume of frozen gizzard myosin to obtain 3-5 mg.

2.Dialyze against buffer 1 overnight to remove residual ammonium sulfate.

3.Clarify myosin solution at 20,000 rpm,4℃,for 30 min in a 50Ti rotor.

4.Dilute 20μl myosin with 400μl dialysis buffer.Determine concentration of myosin by reading OD₂₈₀ (UV).

concentration (mg/ml)= OD * 21 / 0.508

5.Measure volume of myosin.Determine total amount of myosin.

6.Dissolve 0.5-1 mg IATR in 100μl of DMSO; pipet until dye forms a fine slurry.It is very important at this point that the dye be dissolved as much as possible.

7.Add the slurry dropwise to buffer 2 while stirring,to obtain a concentration of 0.1 mg/ml.

8.Clarify dye at 35,000 rpm,4℃,for 15 min in a 50Ti rotor.

9.Carefully collect supernatant.Dilute 100 to 1000 fold and read OD at 555 nm.The volume required for reaction (in ml)is calculated as:(1.4/OD₅₅₅)x mg of myosin

10.Add dye to myosin,mix gently by pipeting.Incubate on ice for 2 hr in the dark.Equilibrate column with buffer 2.

11.Apply to Bio-Beads column.Pool fractions that appear pink in roomlight.

12.Dialyze overnight against buffer 4 to precipitate myosin.

Day 3

Materials

1.2 M KCl,10 mM PIPES,pH 7.0,4℃,250 ml.

2.0.45 M KCl,2 mM Tris-acetate,pH 7.0,4℃,100 ml (injection buffer).

Procedure

1.Pellet myosin at 15,000 rpm,4℃,for 10 min in a SS34 rotor.

2.Soak and resuspend pellet in ~200μl of buffer 1.Handle gently.

3.Clarify at 25,000 rpm,4℃,for 20 min in a 42.2Ti rotor.

4.Determine myosin concentration using Lowry assay.Determine dye concentration by diluting conjugate 1/40 and reading OD at 555 nm.Calculate the dye/protein molar ratio.

D/P = {OD₅₅₅ x 41 / 60,000} / {(mg/ml)/ 470,000}

5.For microinjection,dialyze 2 hr against injection Buffer.

6.Ideal myosin concentration for injection is 7-10 mg/ml.Typical labeling ratio = 4.0-5.0.

7.Gizzard myosin good for ~2 day.Continue to dialyze against the injection buffer until just before use,then clarify.