Principle of the assay: This assay will test specific interactions between proteins and
immobilized lipids. It is essentially most similar to immunoblotting, with the added step of protein
binding preceding the antibody steps.
Special reagents and equipment:
Fatty Acid-Free (FAF) BSA Sigma Cat# A6003
Hybond C extra membrane Amersham Biosciences Cat # RPN2020E
Procedure:
1. Dilute lipids of interest in chloroform to equal concentration such that your desired amount
of lipid can be achieved in a total volume of 5 ul. *
2. Spot 5 ul of each dilution 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 μg/ml in 3% FAF-BSA/TBS-T. Incubate membrane with protein
solution overnight at 4
o
C.*****.
7. Next morning: wash the membrane 6 times (5 minutes for each wash) with TBS-T.
8. Incubate with primary antibody in 3% FAF-BSA/TBS-T for 1 hr. at room temperature (do
not use nonfat dry milk, use the FAF-BSA throughout the entire procedure).******
9. Wash as in 7.
10. Incubate with secondary antibody in 3% FAF-BSA/TBS-T for 1 hr at room temperature.
11. Wash 12 times (5 minutes each wash) with TBS-T.
12. Visualize protein binding using enhanced chemiluminescence. Expose to film for 5
minutes for an initial trial and modify time as needed. .*******