Last updated:  18Feb91 by Chris Hug

1. Start with lyophilized conventional actin (containing 2 g sucrose per g actin).
2. Equilibrate Sephadex G-150 gel filtration column with fresh G-buffer overnight. Dialyze conventional actin vs G-buffer, 10 ml at 6 mg/ml for each G-150 column run planned. Change dialysis once, mixing dialysis tube.
3. Spin 100,000g (40,000 rpm in Ti75, which takes 10 ml tubes) for 2 hours.
4. Take top 7 ml with pasteur pipet, avoiding floating lipids and pelleted actin filaments. Lower 2-3 ml contains actin oligimers and should be avoided.
5. Load 7 ml onto G-150 column; after this has run into the bed, layer on 2 ml G-buffer, being careful not to disturb top of bed. Let run in. Add G-buffer, connect to resevoir. Collect fractions: 70 drops/tube on Isco, 125 drops/tube of Elson's. If on Isco, read chart to determine which fractions to pool; verify by reading at A₂₉₀. If not using chart recorder, read A₂₉₀ and plot.
6. Pool from halfway up second peak, avoiding earlier portion of peak that can contain CapZ. Take A290 of pool. Concentation (mg/ml) = A₂₉₀ / 0.63.
7. Add 2 mg sucrose per mg actin, place in lyophizer flask. Freeze in liquid nitrogen, rotating to make cone. Lyophilze on Biochemistry lyophilizer, second floor Cancer Research. Store -20 degrees in a labelled scintillation vial in jar "Actin- not labelled", lab freezer.
8. Wash column with additional G-buffer and clamp.
9. Keep notes and chart recordings in lab notebook "Actin Preps"