Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells
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Author:
Date:
2018-08-20
[Abstract] Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 μm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer’s group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in ...
[摘要] 纤连蛋白(FN)是一种细胞外基质蛋白,由许多细胞类型分泌,主要与细胞表面受体整合素α5β1结合。整合素α5β1结合启动FN逐步组装成原纤维,这一过程称为原纤维形成。我们和其他几个人已经证明了原纤维形成对细胞迁移和转移的关键作用。虽然免疫染色和显微镜方法有助于可视化FN掺入原纤维,每个原纤维的长度至少为3μm,但是第一项研究开发了一种生物化学分离FN以量化原纤维并入FN的方法,由Jean Schwarzbauer小组于1996年出版。我们的方案改编自原始出版物,并已在多种细胞类型上进行测试,包括如此处所示的MCF10A乳腺上皮细胞和Caki-1肾癌上皮细胞。使用两种洗涤剂提取物,将细胞FN分离成不溶于洗涤剂或掺入原纤维的FN和可溶性FN或未掺入的级分。为了确定原纤维形成是否利用FN的再循环池,我们使用了生物素标记的FN(FN-生物素)再循环测定,其已经从先前的研究中修改。使用再循环测定和脱氧胆酸盐分离方法的组合,可以定量地证明在不同实验条件下细胞中原纤维形成的程度,并确定原纤维形成的FN来源
【背景】 纤连蛋白(FN)是普遍产生的细胞外基质(ECM)组分(Uitto et al。,1989; Mao和Schwarzbauer,2005)。纤连蛋白库是转录产生的,可以通过几种生长因子如TGF-β1增加(Yokoi et al。,2002; Mimura ...
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Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization
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Author:
Date:
2017-11-20
[Abstract] Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and co-associated Flag-tagged protein using Streptavidin MagBeads.
[摘要] Pulldown分析是一种常规的方法来确定蛋白质在体外的相互作用。 用两种不同的标签表达感兴趣的蛋白质可以检测两种标签是否可以通过两种标签之一作为同低聚体复合物来捕获。 该方案基于使用链霉抗生物素蛋白MagBeads的链霉抗生物素蛋白珠捕获生物素化蛋白质和共结合Flag标记蛋白质。 【背景】淀粉样前体蛋白(APP)可以通过其大的胞外结构域及其跨膜结构域形成同型二聚体,在生物学功能中起重要作用。 目前的方案已被用于表征APP跨膜C-末端99个氨基酸片段(C99)的同二聚化(Yan等人,2017)。 该检测的基本原理如图1所示:链霉亲和素包被的MagBeads可以捕获生物素化的蛋白质,这可以拉下相互作用蛋白质,并通过抗FLAG抗体检测。
图1.基于MagBeads的pull-down测定的原理在该特定的方案中,使用生物素化的Avi-标记的C99蛋白和相关的C99-TEV位点-rTA-Flag蛋白质。
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