Lipid-Protein Overlay Assay (Fat Blotting)

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. .*******

Reference:

1. Stevenson, J.M., I.Y. Perera, and W.F. Boss, A phosphatidylinositol 4-kinase pleckstrin

homology domain that binds phosphatidylinositol 4-monophosphate. J Biol Chem, 1998.

273(35): p. 22761-7.

2. Dowler, S., et al., Identification of pleckstrin-homology-domain-containing proteins with novel

phosphoinositide-binding specificities. Biochem J, 2000. 351(Pt 1): p. 19-31.

3. Jones, J.A. and Y.A. Hannun, Tight Binding Inhibition of Protein Phosphatase-1 by

Phosphatidic Acid. SPECIFICITY OF INHIBITION BY THE PHOSPHOLIPID. J Biol Chem,

2002. 277(18): p. 15530-8. Jones JA, Hannun YA.Tight binding inhibition of protein phosphatase-1 by phosphatidic acid.

Specificity of inhibition by the phospholipid. J Biol Chem. 2002 May 3;277(18):15530-8. Epub

2002 Feb 20.

Author: Jeffrey Jones (modified by Bill Wu)

Your email: wub@musc.edu

Date: Dec-2009