Name: lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction
Uploaded: 2011-04-26T16:00:00
Description: Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

这项工作是由美国国立卫生研究院拨款R01NS046835和R01AG034389，三月优生优育基金会授予6FY08 - 322的支持。特别感谢你致力于夫人埃莉诺布朗（美天旎生物技术，赤褐色，CA），极大地帮助她的洞察力到MACS科技。美天旎慷慨地提供实验示范使用，不需任何费用的材料。我也很感谢博士王广沪（医学院格鲁吉亚/格鲁吉亚卫生科学大学，奥古斯塔，佐治亚州）表达细胞株产生的PKCζ。也承认在格鲁吉亚/佐治亚州健康科学大学（下林美博士的董事）医学院分子医学研究所的支持。

1。简介在我们的实验室开发的脂质囊泡介导的亲和层析法，使用磁激活细胞分选（LIMACS）技术隔离酰胺相关的蛋白质复合物1-3。本来，脂质囊泡酰胺和磷脂酰丝氨酸，允许使用磁粒子标记的膜联蛋白V（高度仿射磷脂酰丝氨酸）隔离囊泡及其相关蛋白的Mac。我们已经使用的LIMACS 技术，在体外再造一个酰胺相关的极性复杂，从细胞神经酰胺结合蛋白的分离...

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	<li>Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+binding+to+ceramide+activates+protein+kinase+Czeta+before+the+formation+of+a+pro-apoptotic+complex+with+PAR-4+in+differentiating+stem+cells.">Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells.</a> J Biol Chem. 280, 26415-26424 (2005).</li><li>Wang, G., Krishnamurthy, K., Umapathy, N. S., Verin, A. D., Bieberich, 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+carboxyl-terminal+domain+of+atypical+protein+kinase+Czeta+binds+to+ceramide+and+regulates+junction+formation+in+epithelial+cells.">The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.</a> J Biol Chem. 284, 14469-14475 (2008).</li><li>Chalfant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+ceramide+regulates+the+alternative+splicing+of+caspase+9+and+Bcl-x+in+A549+lung+adenocarcinoma+cells.+Dependence+on+protein+phosphatase-1.">De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.</a> J Biol Chem. 277, 12587-12595 (2002).</li><li>Simon, C. G., Holloway, P. W., Gear, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exchange+of+C(16)-ceramide+between+phospholipid+vesicles.">Exchange of C(16)-ceramide between phospholipid vesicles.</a> Biochemistry. 38, 14676-14682 (1999).</li><li>Goni, F. M., Contreras, F. X., Montes, L. R., Sot, J., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysics+(and+sociology)+of+ceramides.">Biophysics (and sociology) of ceramides.</a> Biochem Soc Symp. , 177-188 (2005).</li><li>Kumagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CERT+mediates+intermembrane+transfer+of+various+molecular+species+of+ceramides.">CERT mediates intermembrane transfer of various molecular species of ceramides.</a> J Biol Chem. 280, 6488-6495 (2005).</li><li>Wessel, D., Flugge, U. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+quantitative+recovery+of+protein+in+dilute+solution+in+the+presence+of+detergents+and+lipids.">A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.</a> Anal Biochem. 138, 141-143 (1984).</li></ol>

要测试的脂质和蛋白之间的相互作用，是阻碍嵌入细胞膜的脂质。细胞膜由一些脂类和蛋白质的混合​​物，它是在组织脂质微区或木筏。因此，微区和蛋白质的合作，净化不能明确区分，如果一种蛋白质直接结合到脂质，或仅仅是在微区结构丰富。使用涂层表面上，如脂质酶联免疫吸附或等离子体共振光谱的定义血脂的其他方法可以检测其结合蛋白的一个特定的脂质相互作用..然而，在一个有?...

Annexin V的共轭磁珠和Macs和微观minicolumns提供了由美天旎生物技术公司（赤褐色，CA）。血脂最高纯度和阿凡提极性脂（雪花石膏，AL）获得。

lipid vesicle-mediated affinity chromatography using magnetic activated cell sorting (limacs): a novel method to analyze protein-lipid interaction

脂蛋白质相互作用的分析是困难的，因为嵌在细胞膜的脂质，因此，无法以最净化程序。血脂作为一种替代方法，可以在平坦的表面涂层，为脂质ELISA法和等离子体共振光谱。然而，表面涂层血脂不形成微区结构，这可能是重要的脂质绑定属性。此外，这些方法不容许较大数额的蛋白结合他们的目标血脂净化。 为了克服这些限制测试脂蛋白质相互作用和净化脂质结合蛋白，我们开发了一种新的方法称为脂质囊泡介导的亲和层析法，使用磁激活细胞分选（LIMACS）。在此方法中，脂质囊泡锚脂质膜联蛋白V的Mac与目标脂质和磷脂酰准备。磷脂是一个无处不在的细胞膜磷脂，高亲和力的蛋白膜联蛋白V。使用Annexin V的共轭含有的磷脂脂质囊泡结合磁珠磁珠。当脂质囊泡与细胞培养裂解的蛋白结合的目标脂质也将受到约束珠，可以共同使用Mac纯化。这种方法也可以用来测试一个目标脂质蛋白复合物结合，如果重组蛋白重组。 我们已经用这种方法，以显示与鞘脂酰胺和合作净化前列腺凋亡反应4（4杆），蛋白结合酰胺相关aPKC非典型蛋白激酶C（aPKC）的相互作用。我们也使用这种方法，重组的重组aPKC神经酰胺与细胞极性相关的蛋白质Par6和Cdc42复杂。由于脂质囊泡可以与各种sphingo或磷脂准备，LIMACS脂质 - 蛋白相互作用密切类似于细胞膜的脂质环境提供了通用的测试。其他脂质蛋白复合物可以识别使用的脂质结合蛋白与脂质囊泡的合作纯化的蛋白质组学分析。

Watch this Scientific Journal Video about Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction at JoVE.com

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

为了测试我们使用Macs和Annexin V的共轭磁珠和目标脂质和Annexin V的具有约束力的磷脂酰丝氨酸合成脂质囊泡与目标脂质的一种蛋白质的相互作用。绑定到目标脂质蛋白质纯化和分析后，从珠洗脱。

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Research

JoVE Journal

Biology

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

Fluorescence Activated Cell Sorting of Plant Protoplasts

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

科研

生物学

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

结合母细胞和波动测试磁分选，以分析基因组不稳定性的过程中有丝分裂细胞衰老<em&gt;酿酒酵母</em

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

使用串联亲和纯化的MyoD相互作用组鉴定质谱分析法

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

本机配合物的净化对结构研究使用串联亲和标签法

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...