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. In this technical note, we share several specific improvements to ensure lipid-protein overlay assays are of high quality and contain proper controls.

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. Fluorescently labeled lipids have been a useful tool to understand the membrane behavior properties of lipids such as phosphatidylserine, cholesterol, and some phosphoinositides. However, to the best of our knowledge, fluorescently labeled C1P hasn't been implemented to investigate its ability to serve as a mimetic of endogenous C1P in cells or untagged C1P in in vitro experiments. Cellular and in vitro assays demonstrate TopFluor-C1P harbors a fluorescent group that is fully buried in the hydrocarbon core and fluoresces across the spectrum of physiological pH values. Moreover, TopFluor-C1P didn't affect cellular toxicity at concentrations employed, was as effective as unlabeled C1P in recruiting an established protein effector to intracellular membranes, and its subcellular localization recapitulated what is known for endogenous C1P. Notably, the diffusion coefficient of TopFluor-C1P was slower than that of TopFluor-phosphatidylserine or TopFluor-cholesterol in the plasma membrane and similar to that of other fluorescently labeled sphingolipids including ceramide and sphingomyelin. These studies demonstrate that TopFluor-C1P should be a reliable mimetic of C1P to study C1P membrane biophysical properties and C1P interactions with proteins.