Gel overlay assays
These are a refinement of the western blot technique for studying protein-protein interactions. A specific protein is used as a probe. This is chemically treated to make a tag (e.g. DIG - digoxigen). The tag is found with an antibody and the antibody detected as for a western blot.
An example
The 14-3-3 proteins have been shown to accumulate in barley-powdery mildew interactions. The are known to bind other proteins in other plants (and animals). Do they bind something in barley? Panel A is stained for protein. Panels C and E show western blots from (a) total leaf (b) leaf epidermis (where the fungus lives) showing the presence of a protein (H+ATPase) in inoculated not uninoculated epidermis. Panels B and D are identical membranes to C and E which have been probed with DIG-14-3-3 protein as described in the figure above. Thus it seems very likely that 14-3-3 is binding the H+ATPase in the inoculated epidermis, confirming yeast two-hybrid results.
(unpublished results)
