Labeling Muscle Actin with 5-iodoacetamidofluorescein
Day 0 and 1
Materials
1. 0.5 mM ATP, 0.2 mM CaCl2, 2 mM Tris-HCl, pH 8.0 at 4oC, 250 ml for day 0.
2. 100 mM KCl, 100 mM boric acid, pH 8.4 at 4oC, 10 ml.
3. 0.5 mM ATP, 2 mM MgCl2, 100 mM KCl, 2 mM DTT, 2 mM PIPES, pH 7.0, 1000 ml.
4. IAF (Molecular probes)
5. 50Ti tubes, small vial.
Procedure (perform under reduced light, 4oC 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 2.4 mg IAF in a test tube. Add 100-200 µl acetone and make a fine slurry by grinding particles against the tube with a pipet. Adjust the volume in step 6 if the amount actually weighed is higher.
6. Add IAF slurry very slowly by dipping a small amount at the tip of a Pasteur pipet into 2 ml of buffer 2 in a small vial under constant stirring.
7. Add 2 ml of the dye solution to actin and mix gently with a Pasteur pipte.
8. Let sit on ice for 2 hr.
9. Stop the reaction by adding DTT to 10 mM.
10. Dialyze against 1 liter buffer 3 overnight.
Day 2
Materials
1. Buffer 1 as for day 1, 4oC, 2000 ml.
2. G-25-150 column, ~30x1.5 cm.
3. 50Ti tubes, volumetric conical tube.
4. Colloidin bag for dialysis.
Procedure
1. Equilibrate G-25 column with Buffer 1.
2. Pellet actin in a 50Ti rotor at 40,000 rpm, 4oC, for 2 hr.
3. Rinse pellet briefly with buffer 1, soak and resuspend in 2 ml.
4. Dialyze against 500 ml buffer 1 at 4oC for 4-5 hr in a colloidin bag.
5. Clarify dialyzed actin in a 50Ti rotor for 1 hr at 40,000 rpm, 4oC.
6. Run supernatant through the G-25 column, collect 10 drop fractions.
7. Collect fluorescent fractions in the void volume, measure volume in a volumetric conical tube.
8. Polymerize actin by adding KCl to 100 mM and MgCl2 to 2 mM. Let sit for 30-60 min at room temperature.
9. Centrifuge in a 50Ti rotor for 2 hr at 40,000 rpm, 15oC.
10. Soak pellet(s) in 0.4 ml buffer 1 for 1-2 hr, resuspend by gentle pipeting.
11. 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 4oC.
2. 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}, should be 0.5-0.9.
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.
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