Protein-Lipid Overlay Assay

from Emr Lab via Lois Weisman

1. Prepare serial two-fold dilutions of PIP starting from 0.2mM dissolved in a resuspension solution of CHCl3:CH3OH:50mM HCl (5:10:4, v/v/v) with 2ml Ponceau S (Fluka 09276).
2. Cut the proper size of nitrocellulose membrane (GE Water & Process Technologies, WP4HY00010).
3. Spot 1ml of the PIP solution on the membrane.
4. After drying blots in dark at room temperature (1 hr) & at 4oC (overnight), incubate the blot for 1hr at room temperature in a blocking solution (5% non-fat dry milk in TBS with 0.1% Tween20).
5. Then incubate the blot overnight at 4oC with 5mg/ml the bacterial expressed and purified GST-fusion proteins in 0.5% fatty-acid free BSA (Sigma) in TBS with 0.1% Tween20.
6. After washing 5 times each 5 minutes with TBS with 0.1% Tween20, incubate the blot overnight in a cold room with anti-GST antibody in a solution of 0.5% fatty-acid free BSA (Sigma) in TBS with 0.1% Tween20.
7. Wash the blots and incubate for 1hr at room temperature with a secondary antibody in 5% non-fat dry milk in TBS with 0.1% Tween20.
8. Wash as in (5) & visualize the blot using Super-Signal chemiluminescent kit (Pierce).