PLiMAP:基于接近度的膜相关蛋白标记

Q1 Biochemistry, Genetics and Molecular Biology

Current Protocols in Protein Science Pub Date : 2020-06-30 DOI:10.1002/cpps.110

Gregor P. Jose, Thomas J. Pucadyil

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引用次数: 6

摘要

外周膜蛋白参与多种生物学途径。因此，分析其膜结合特性的方法变得非常重要。在本报告中，我们详细介绍了一种光活化荧光脂质的合成和利用方案，作为监测蛋白质膜结合的报告。该检测被称为基于接近度的膜相关蛋白标记(PLiMAP)，是基于紫外线激活的荧光脂质报告蛋白，该报告蛋白反过来与结合在膜上的蛋白质交联并使其荧光。©2020 Wiley期刊公司。基本方案1:合成bodipy -二嗪磷脂酰乙醇胺(BDPE)基本方案2:制备含BDPE的脂质体基本方案3:用候选蛋白进行PLiMAP基本方案4:通过分析凝胶内荧光定量候选蛋白的脂质体结合特性支持方案:纯化SidM蛋白的GST-2×P4M结构域

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PLiMAP: Proximity-Based Labeling of Membrane-Associated Proteins

Peripheral membrane proteins participate in numerous biological pathways. Thus, methods to analyze their membrane-binding characteristics have become important. In this report, we detail protocols for the synthesis and utilization of a photoactivable fluorescent lipid as a reporter to monitor membrane binding of proteins. The assay, referred to as proximity-based labeling of membrane-associated proteins (PLiMAP), is based on UV activation of a fluorescent lipid reporter, which in turn crosslinks with proteins bound to membranes and renders them fluorescent. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis of BODIPY-diazirine phosphatidylethanolamine (BDPE)

Basic Protocol 2: Preparation of BDPE-containing liposomes

Basic Protocol 3: Performing PLiMAP with a candidate protein

Basic Protocol 4: Quantitation of liposome-binding properties of the candidate protein from analyzing in-gel fluorescence

Support Protocol: Purification of GST-2×P4M domain of SidM protein

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来源期刊

Current Protocols in Protein Science Biochemistry, Genetics and Molecular Biology-Biochemistry

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期刊介绍： With the mapping of the human genome, more and more researchers are exploring protein structures and functions in living organisms. Current Protocols in Protein Science provides protein scientists, biochemists, molecular biologists, geneticists, and others with the first comprehensive suite of protocols for this growing field.