Notes and tips for improving quality of lipid-protein overlay assays.

To reduce costs of lipid-binding assays, allow for multiple lipids to be screened for protein binding simultaneously, and to make lipid binding more user friendly, lipids have been dotted onto membranes to investigate lipid-protein interactions. These assays are similar to a western blot where the membrane is blocked, incubated with a protein of interest and detected using antibodies. Although the assay is inexpensive and straightforward, problems with promiscuous or poor binding, as well as insufficient blocking occur frequently.

Investigation of the biophysical properties of a fluorescently modified ceramide-1-phosphate.

Ceramide-1-phosphate (C1P) is an important signaling sphingolipid and a metabolite of ceramide. C1P contains an anionic phosphomonoester head group and has been shown to regulate physiological and pathophysiological processes such as cell proliferation, inflammation, apoptosis, phagocytosis, and macrophage chemotaxis. Despite this mechanistic information on its role in intra- and intercellular communication, little information is available on the biophysical properties of C1P in biological membranes and how it interacts with effector proteins.