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Actin Capture Assay

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.
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