G-150 Actin Prep

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"