- Dialyze purified GST fusion proteins and actin into PBS + 1mM MgCl2.
- Mix 5ug actin into 50ul total volume binding buffer.
- Mix 5ug GST-fusion protein into total volume 50ul binding buffer.
- Combine actin + GST-fusion protein and incubate for 1 hr. x 4¡C.
- Add 20ul 50% glutathione agarose equilibrated into binding buffer. Incubate 15 min x 4¡C with frequent mixing.
- 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.
- Add 50ul reducing sample buffer, heat 2 min. x 95¡C and load 10ul onto a 10% SDS-PAGE.
- 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.