Imagen no invasiva de fosfoinositidos con biosensore de lípidos genéticamente codificados

The localization and metabolism of lipids are difficult to dynamically resolve. Whereas immunofluorescence techniques rely on fixing cells and subsequent postmortem examination, and the employment of fluorescent lipid analogs in turn depends on approximative probes, genetically encoded lipid biosensors are not invasive and report on endogenous targets in living cells. Here we outline a protocol for imaging PI(4,5)P₂ localization in live HEK293A cells by expression of the genetically encoded lipid biosensor PH-PLCδ1. The protocol is presented as having two branching paths, with the first detailing how to overexpress the biosensor for confocal microscopy. The second path describes expression of the biosensor at single molecule levels for subsequent TIRF microscopy.