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.
