| Liposome Flotation Assays for Phosphoinositide-protein Interaction
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Author:
Date:
2017-03-05
[Abstract] Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different subcellular compartments (Di Paolo and De Camilli, 2006; Lemmon, 2003). Therefore, analysis of these effectors’ binding properties and specificity towards different phosphoinositides is crucial for the understanding of their cellular functions. This protocol describes a method to characterize the binding of proteins to different phosphoinositide-containing vesicles.
[摘要] 磷酸肌醇是涉及控制主要细胞功能和信号通路的稀有膜脂质。他们能够将特异性效应物蛋白募集到质膜和细胞器的胞质表面，以协调各种信号传递和转运过程，并保持不同亚细胞区室的特定身份（Di Paolo和De Camilli，2006; Lemmon， 2003）。因此，这些效应物对不同磷酸肌醇的结合特性和特异性的分析对于了解其细胞功能至关重要。该方案描述了表征蛋白质与不同含磷酸肌醇的囊泡结合的方法。
背景 分析蛋白质 - 磷酸肌醇结合和对磷酸肌醇家族不同成员的特异性的几种方法：脂质覆盖，脂质浮选测定和表面等离子体共振（SPR）。脂质浮选测定法包括将单层囊泡与目的蛋白质孵育，随后在蔗糖垫上浮选囊泡，其将单独的囊泡结合的蛋白质与未结合的蛋白质和囊泡分离。与其他方法相比，脂质浮选测定是i）在技术上比SPR更容易和更便宜，ii）与蛋白质重叠测定相比，更具体和更接近于生理条件，因为它模拟膜的曲率，其中脂质被干燥平的硝酸纤维素膜。该方案描述了重组GST标记的蛋白质与确定大小和脂质组成的单层囊泡的结合，特别是关于对不同单磷酸化磷酸肌醇（PIP）的特异性。
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| PTEN-lipid Binding Assay
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Author:
Date:
2013-08-20
[Abstract] The lipid and protein interactions are an integral and important part of many cellular signaling pathways. The understanding of the selective and specific interaction of the given lipid molecule with the target protein is required for studying cellular signaling. In this assay, different lipids are spotted onto a nitrocellulose membrane to which they attach. Then the membrane is incubated with a lipid binding protein possessing an epitope tag. The protein binds to the lipid which is detected by immunoblotting with an antibody recognizing the epitope tag (see Figure 1). PTEN is an important tumor suppressor which functions as both protein and lipid phosphatase. The primary physiological substrate of PTEN is signaling lipid PtdIns (3, 4, 5) P3, by dephosphrylating PtdIns (3, 4, 5) P3 to ...
[摘要] 脂质和蛋白质相互作用是许多细胞信号传导途径的整体和重要部分。研究细胞信号传导需要理解给定脂质分子与靶蛋白的选择性和特异性相互作用。在该测定中，将不同的脂质点在它们附着的硝酸纤维素膜上。然后将膜与具有表位标签的脂质结合蛋白一起温育。蛋白质与脂质结合，其通过用识别表位标签的抗体的免疫印迹检测（参见图1）。 PTEN是一种重要的肿瘤抑制剂，其作为蛋白质和脂质磷酸酶起作用。 PTEN的主要生理学底物是信号传导脂质PtdIns（3,4,5）P3，通过将PtdIns（3,4,5）P3去磷酸化为PtdIns（4,5）P2PTEN负调节PI3K信号传导并介导其肿瘤抑制功能通过灭活下游致癌AKT介导的信号。进行PTEN脂质结合测定以研究PTEN与不同脂质分子的特异性结合。
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| RhoGTPase Activation Assay
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Author:
Date:
2012-10-05
[Abstract] This protocol describes procedures to assay for GTP-bound (active) form of small GTPases of the Rho superfamily in human brain cancer (glioma) cell lines but can also be applied to cells or tissues of other origins. The principle of assay is based on the property of these active GTPases to interact with their specific effectors (e.g. Rhotekin for RhoA; p21-activated kinase (PAK) for Rac1 and Cdc42. In essence, Rhotekin or PAK1 are expressed in the form of GST-fusion protein to pull down the corresponding active GTPases.
[摘要] 属于黄病毒科的JEV（ 病毒）与CLEC5A（C型凝集素结构域家族5，成员A） C型凝集素，与DAP12信号传导蛋白相关并且在骨髓细胞上表达，与登革热病毒的程度相同。 该方案用于通过ELISA方法进行和确定纯化的JEV颗粒与人和鼠CLEC5A.Fc融合蛋白之间的相互作用。 不同的菌株和批次的纯化JEV以及CLEC5A.Fc融合蛋白的纯度可能影响吸光度的结果。 如果用户打算进行ELISA测定，则很可能需要修饰。...
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