Quantifying the Proteolytic Cleavage ofPlasma Membrane Proteins in LivingCells

Martino Calamai; Francesco Saverio Pavone

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Quantifying the Proteolytic Cleavage ofPlasma Membrane Proteins in LivingCells

机译：量化活细胞中血浆膜蛋白的蛋白水解裂解

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The standard approach to study the activity of proteases consists of lysing cellsand measuring the changes in the fluorescence properties of a synthetic substrateafter cleavage in vitro. Here, a general protocol that uses a bi-fluorescentchimeric construct of a known substrate protein that follows the proteolyticprocessing in living cells is described. This approach is useful, in particular,to search for pharmacological conditions altering the cleavage rate of a certainprotease, or to investigate the biological factors influencing a certain proteolyticmechanism. Three different methods (microscopy, flow cytometry, andspectroscopy) to detect fluorescence changes due to alteration in the processingare described. This approach was originally developed for studying conditionsaffecting the proteolytic activity of the β-secretase Bace1 on the amyloid precursorprotein APP, but can in principle be applied to investigate any membraneprotein undergoing ectodomain shedding by proteolytic cleavage.

机译：研究蛋白酶活性的标准方法包括裂解细胞和测量体外合成亚样品切割的荧光特性的变化。这里，描述了使用遵循活细胞中蛋白水解性的已知底物蛋白的双荧光Chim构建体的一般方案。该方法尤其是用于寻找改变某种蛋白酶的切割率的药理学条件，或者研究影响某种蛋白水解机制的生物因素。由于所描述的处理方法中描述的改变，三种不同的方法（显微镜，流式细胞术，和光谱）以检测荧光变化。该方法最初开发用于研究淀粉样蛋白前体蛋白应用β-分泌酶Bace1的蛋白水解活性的方法，但原则上可以应用通过蛋白水解裂解进行癌症脱落的任何膜普选蛋白。

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来源
《Current Protocols in Cell Biology》 |2018年第1期|共11页
作者
Martino Calamai; Francesco Saverio Pavone;
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作者单位

National Institute of Optics National Research Council of Italy (CNR) Florence Italy;

LENS - European Laboratory for Non-linear Spectroscopy Florence Italy;

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原文格式 PDF
正文语种 eng
中图分类细胞生物学;
关键词
flow cytometry; fluorescence; imaging; proteolysis; spectrofluorimetry;

机译：流式细胞术;荧光;成像;蛋白水解;光谱氟状物;

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1. Quantifying the Proteolytic Cleavage ofPlasma Membrane Proteins in LivingCells [J] . Martino Calamai, Francesco Saverio Pavone Current Protocols in Cell Biology . 2018,第1期

机译：量化活细胞中血浆膜蛋白的蛋白水解裂解
2. Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins. [J] . Pham VC, Henzel WJ, Lill JR Electrophoresis: The Official Journal of the International Electrophoresis Society . 2005,第22期

机译：快速的膜上蛋白水解切割，用于埃德曼测序和蛋白质的质谱鉴定。
3. Protein Quantification by Isotope Dilution Mass Spectrometry of Proteolytic Fragments:Cleavage Rate and Accuracy [J] . Cristian G.Arsene, Rudiger Ohlendorf, William Burkitt Analytical chemistry . 2008,第11期

机译：通过蛋白水解片段的同位素稀释质谱对蛋白质进行定量：切割速率和准确性
4. A Sensitive and Reproducible Workflow for Quantification of Membrane-associated Proteins Using Liquid Chromatography Coupled Targeted Mass Spectrometry [C] . Meiyao Wang, Hua-Jun He, Gun-Young Heo, American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics . 2013

机译：使用液相色谱偶联靶标质谱法定量膜相关蛋白的敏感和可再现的工作流程
5. Proteolytic mechanisms that degrade the transmembrane domain of membrane proteins and the relationship to the intramembrane cleavage by gamma-secretase in Alzheimer's disease. [D] . Bunnell, William Lewis. 1998

机译：在阿尔茨海默氏病中，降解膜蛋白跨膜结构域的蛋白水解机制及其与γ-分泌酶与膜内裂解的关系。
6. Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion. [O] . M F Frana, J N Behnke, L S Sturman, 1985

机译：鼠冠状病毒E2糖蛋白的蛋白水解裂解：蛋白水解裂解和细胞融合的宿主依赖性差异。
7. Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis [O] . Susumu Uehara, Ayane Sei, Misaki Sada, 2020

机译：通过拟南芥中的嵌合蛋白的蛋白水解裂解来实现正宗的BICA和SBTA蛋白的安装到叶绿体包膜膜中

Quantifying the Proteolytic Cleavage ofPlasma Membrane Proteins in LivingCells

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