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Actin Capture Assay

David Amberg

  1. Dialyze purified GST fusion proteins and actin into PBS + 1mM MgCl2.
  2. Mix 5ug actin into 50ul total volume binding buffer.
  3. Mix 5ug GST-fusion protein into total volume 50ul binding buffer.
  4. Combine actin + GST-fusion protein and incubate for 1 hr. x 4¡C.
  5. Add 20ul 50% glutathione agarose equilibrated into binding buffer. Incubate 15 min x 4¡C with frequent mixing.
  6. Wash beads 4 times with binding buffer, spin down 4-8000 rpm for 30 seconds. On the last wash transfer to a new tube, spin down and remove as much liquid as possible.
  7. Add 50ul reducing sample buffer, heat 2 min. x 95¡C and load 10ul onto a 10% SDS-PAGE.
  8. Execute western according to my protocol. For primary antibody use anti-GST at 1:1000 and anti-actin at 1:1000. For secondary use HRP conjugated IgG at 1:10,000.

Actin Capture Assay Reagants

  • Binding Buffer: 1X PBS pH 7.2 (Lane Recipe) + 10% Glycerol + 1mMMgCl2 + 0.2mM ATP + 0.1% BSA + 0.01% Triton X-100 + 1mM DTT + 1mM EGTA. Variables include lowering salt (above recipe is about 136mM in NaCl). replacing EGTA with 1mM CaCl2, replacing ATP with ADP.
  • ATP Sigma#A5394
  • ADP Sigma #A6521
  • 10X PBS: 80 grams NaCl + 2 grams KCl + 14.4 grams Na2HPO4 + 2.4 grams KH2PO4. pH upon dilution to 1X should be 7.2.
  • BSA Sigma #A7638.
  • Glutathione-Agarose Sigma# G-4510. Prepare according to instructions.
  • Anti-GST Molecular Probes, Inc. #A5800. Rabbit IgG (H+L) fraction.
  • Peroxidase conjugated Protein A Cappel #55901.
  • Guinea Pig anti-actin Animal #2 whole serum, Botstein lab, prepared by Jon Mulholland.
  • Sample Buffer: 1ml 0.5M TRIS pH6.8 + 4.4ml ddH2O + 2.0ml 10% SDS + 2.0ml glycerol + 1.0ml 1M DTT.