Michael Worcester, Magdalene Motter, Gerald R V Hammond
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引用次数: 0
Abstract
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.
期刊介绍：
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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