1. Follow usual procedure for actin prep up to the first high speed spin to separate troposmyosin, etc.
If you already have purified actin, just polymerize in buffer A with 20x salts to a concentration of 5-10 mg/ml and proceed to step 3.
The amount of pyrene to add is for about 100 mg of actin. For less amount decrease the amount of label.
2. Suspend the pellet from first high speed spin in 40 ml of the following buffer: 10 mM Hepes, 2 mM MgCl2, 100 mM KCl, 0.5 mM ATP
3. Add 400 µl of 14 g/l pyrene (PIA) (in dimethyl formamide or DMSO) while vortexing the suspension on low speed, because the label is not too soluble in water and the DMF or DMSO may denature some of the protein before it gets diluted to 1%.
4. Let solution sit in foil-wrapped container in dark at 4oC overnight.
5. Spin solution 10 min @ 10k rpm (lower speed is ok), 4oC, to remove undissolved pyrene and denatured actin. Collect the supernatant in TLA.3 rotor ultracentrifuge tubes.
6. Spin for 1h @ 80k rpm, 4oC to pellet F-actin.
7. Resuspend pellet in 10-15 ml of buffer A(G) in douncer. Repeat low speed spin if necessary.
8. Dialyze in 4 L of buffer A(G) overnight and at least one more complete day with daily changes of buffer A(G).
9. Spin 1 h at 80,000rpm, 4oC.
10. Measure absorbance at 290 nm and 344 nm and determine concentrations and
% labeling using
[Pyrene] = A344 / 22,000 (M^-1)
[Actin] = (A290 - (A344 * 0.127)) / 26,600 (M^-1)
11. Divide supernatant into aliquots and freeze in nitrogen.
1 L of 10X Hepes Buffer: 23.83 g Hepes, 50 ml 1 M MgCl2, 74.55 g KCl, pH 7.4
Add ATP to 1X buffer immediately before use.