Lipid-Protein Overlay Assay (Fat Blotting)

This assay will test specific interactions between proteins and lipids. It is essentially most similar to immunoblotting, with the added step of protein binding proceeding the antibody steps. Pay particularly close attention to the number of washes. These blots require substantial washing, error on the side of washing too much instead of too little. This example probes the interaction between 6XHis-PP1cg and a panel of phospholipids.

1. Dilute lipids of interest into chloroform such that you desired concentration can be achieved in a total of 5 ul.

2. Spot 5 ul amounts of lipid on to Hybond C extra membrane (mixed ester supported nitrocellulose).

3. Allow the membrane to dry at room temperature for at least 1 hr post-spotting.

4. Wet the membrane by floating on nano-purified water for 10 minutes, followed by equilibration in buffer for 5 minutes (TBS-T (0.1% Tween-20)).

5. Block membrane in 3% FAF-BSA/TBS-T for 1 hr at room temperature.

6. Dilute your protein to 0.2 ug/ml in 3% FAF-BSA/TBS-T. Incubate membrane with protein solution overnight at 4oC.

7. Next morning: wash the membrane 6 times over 30 minutes with TBS-T

8. Incubate with Primary Antibody: 1:2000 dilution of anti-6XHis (Clonetech) in 3% FAF-BSA/TBS-T for 1 hr. at room temperature (this can probably be done using 5% non-fat dry milk as well).

9. Wash as in 7.

10. Incubate with Secondary Antibody: 1:4000 dilution of goat anti-mouse peroxidase (Jackson ImmunoResearch) in 3% FAF-BSA/TBS-T for 1 hr at room temperature (again can probably be done with 5% nfdm/TBS-T).

11. Wash 12 times over 1 hr with TBS-T.

12. Visualize protein binding using enhanced chemiluminescence. Expose to BioMax film for 0.5-20.0 minutes.