LIPID-PROTEIN OVERLAY ASSAY (from Simon Dowler, Alessi lab.)

Lipids

Dissolve the lyophilised phosphatidylinositol lipids in

MeOH:chloroform:water (in a 2:1:0.8 ratio). Dilute the stock solution in the same solvent prior to use. Be very careful to seal containers containing these lipids properly, otherwise the stock will not be at a reliable concentration. Lipid stocks are stored in the -80 freezer.

Assay

Spot 1µl aliquots of lipid on to Hybond-C extra membrane (Amersham Life Sciences) and air dry for 1hr at room temp. A dilution series of the lipid will give you a better idea of the lipid binding specificity of your protein. Typically, 1.0-500pmole will allow you to see low and high affinity binding.Membranes and Hamilton syringes are stored above the HPLC.

Block the membrane in 3% (w/v) BSA / 50mM Tris/HCl pH7.5, 150mM NaCl, 1mM MgCl₂ and 0.1% Tween-20 for 1hr at room temp. NOTE: higher concentrations of Tween-20 strip the lipids from the membrane.

Incubate membrane with 0.2-1µg/ml purified GST-fusion protein diluted in blocking buffer o/n at 4°C on a rocker.

Wash the membrane 4 x 15min with TBS/1mM MgCl₂-0.1%Tween-20 at room temp.

Detect protein bound to the lipids with an HRP coupled-GST antibody at a 1:5000

dilution in the blocking buffer (we use SIGMA catalogue no. A7340-there should be a working aliquot in the fridge and other aliquots are stored at -20).

Incubate at room temp for 1hr.

Wash as above, with the last wash without Tween.

Detect with enhanced chemiluminescence reagent.

Problems

MAIN PROBLEM IS HIGH BACKGROUND

• Reduce concentration of protein

• Change washing times e.g. 6 x 5mins

• If the protein is epitope tagged or you have specific antibodies against the protein, try these instead. The GST-antibody is generally dirtier with BSA compared with milk

• Check the activity of your protein (if you can) to check that the protein

isn't denatured.

• Try milk to block the membranes. This does give cleaner backgrounds but, I'm not sure whether it removes some of the lipids, or not.