Name: measuring plasma membrane protein endocytic rates by reversible biotinylation
Uploaded: 2009-12-23T16:00:00
Description: Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

这项工作是由美国国立卫生研究院授予＃DA15169到下摆

程序概述： 使用这种方法，细胞表面的蛋白质共价键上标有使用贩运的限制性条件（即低温）下一个膜impermeant，二硫化碳耦合生物素试剂（磺酸基- NHS - SS -生物素）外赖氨酸残基与生物素（见图1插图）。一个细胞转移到贩卖宽松的条件下（37℃）和生物素化蛋白的内化。其他细胞保持在较低的温度控制为1）总时间= 0的表面蛋白，和2）剥离控制。一化后的短期内，细胞移回低?...

<ol>
	<li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat Neurosci. 8, 881-888 (2005).</li><li>Boudanova, E., Navaroli, D. M., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+decreases+in+dopamine+transporter+surface+expression+are+protein+kinase+C-independent.">Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent.</a> Neuropharmacology. 54, 605-612 (2008).</li><li>Boudanova, E., Navaroli, D. M., Stevens, Z., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+transporter+endocytic+determinants:+carboxy+terminal+residues+critical+for+basal+and+PKC-stimulated+internalization.">Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization.</a> Mol Cell Neurosci. 39, 211-217 (2008).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J Biol Chem. 278, 22168-22174 (2003).</li></ol>

常见问题 ：在这些实验中出现的最常见的问题是贫困地带效率。地带的效率是能够解释结果的关键。除非地带高效，是不可能得出结论，任何生物素化蛋白的内化车道，事实上，从表面上看内在​​。带≥90％的效率是最优的，和我们放弃任何结果，如果低于这个水平的地带。一个贫困地带的一个例子是如图3所示。请注意，在加沙地带车道的乐队是非常明显的，并对应一个34％的地带效率?...

measuring plasma membrane protein endocytic rates by reversible biotinylation

质膜蛋白是一种大型的，不同群体的蛋白质受体，离子通道，运输和泵组成。这些蛋白质的活性是负责各种关键的细胞活动，包括营养输送，细胞的兴奋性，和化学信号。许多质膜蛋白质吞贩运，调节通过改变蛋白的表面表达的蛋白质的功能是动态监管。促进蛋白质的内吞作用的机制，是复杂的，并不完全理解许多膜蛋白。为了充分了解的机制，控制某一特定蛋白质的内吞贩运，它是至关重要的蛋白S吞率精确测量。对于许多受体，直接吞率测量经常利用标记的受体的配体。然而，对于许多类，如运输，泵和离子通道的膜蛋白，有没有方便的配体，可以用来衡量吞率。在本报告中，我们描述了一个可逆的生物素的方法，我们聘请来衡量的多巴胺转运蛋白（DAT）的内吞率。这种方法提供了一个简单的方法来测量的内化率，并且可以很容易地雇用贩运大多数膜蛋白研究。

Watch this Scientific Journal Video about Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation at JoVE.com

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

规管的内吞作用管辖的大部分膜蛋白在细胞表面的表达水平。在这里，我们利用还原，膜impermeant生物素化试剂的多巴胺转运蛋白（DAT），一个多面体的膜蛋白的内吞率来衡量的。该方法有利于一个简单的方法来测量大多数等离子的膜蛋白的内吞率。

衡量可逆的生物素的血浆膜蛋白吞价格

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Research

JoVE Journal

Biology

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科研

生物学

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Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

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Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

测绘在细胞膜分子扩散，多目标跟踪（MTT）

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

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Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

视黄醇运输的血浆视黄醇结合蛋白的膜受体的实时分析

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Imaging Plasma Membrane Deformations With pTIRFM

成像质膜变形随着pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

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The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

膜运输过程由单一的蛋白质解析一个高度并行的纳米孔芯片分析系统

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

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This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...