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Diethylenetriaminepentaacetic acid


Company: Sigma-Aldrich
Catalog#: D6518
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Detection of Intracellular Reduced (Catalytically Active) SHP-1 and Analyses of Catalytically Inactive SHP-1 after Oxidation by Pervanadate or H2O2
[Abstract]  Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a ‘signature’ cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013). To detect catalytically active PTPs, cell lysates are treated with iodoacetyl-polyethylene glycol-biotin (IAP-biotin), which irreversibly binds to reduced (S-) cysteine thiols. Irreversible oxidation of SHP-1 after treatment of cells with ... [摘要]  细胞活性氧(ROS)对半胱氨酸依赖性蛋白酪氨酸磷酸酶(PTP)的氧化失活在调节多种细胞类型的信号转导中起关键作用。大多数PTP的磷酸酶活性取决于催化结构域内的“标记”半胱氨酸残基,其在生理pH下保持质子化状态,使其易受ROS介导的氧化。已经开发了用于检测PTP氧化的直接和间接技术(Karisch和Neel,2013)。为了检测催化活性的PTP,用碘乙酰 - 聚乙二醇 - 生物素(IAP-生物素)处理细胞裂解物,所述碘乙酰 - 聚乙二醇 - 生物素(IAP-生物素)不可逆地结合还原的(S-5)半胱氨酸硫醇。使用对磺酸(SO 3)特异性的抗体检测用过钒酸盐或H 2 O 2 2处理细胞后SHP-1的不可逆氧化, H)形式的PTP的保守的活性位点半胱氨酸。在该协议中,我们描述了用于检测造血PTP SHP的还原(S ; active)或不可逆氧化(SO 3 H;非活性)形式的方法-1,尽管这种方法适用于任何细胞类型中的任何半胱氨酸依赖性PTP。

【背景】活性氧(ROS)由细胞NADPH氧化酶和线粒体产生。大多数蛋白质酪氨酸磷酸酶(PTP)含有保守的催化半胱氨酸,其具有低的解离常数(pKa),其对ROS的氧化非常敏感(Rudyk和Eaton,2014)。 PTP的ROS失活在许多细胞类型中调节酪氨酸激酶介导的信号传导反应中起重要作用。在用ROS H 2 O ...

Quantitative Analysis of Cellular Diacylglycerol Content
[Abstract]  Diacylglycerol (DAG) is a bioactive lipid with diverse biological roles. DAG transiently accumulates in a membrane upon receipt of an appropriate stimulus that activates phospholipase C to cleave phospholipids. The resulting hydrolysis product DAG binds to proteins such as protein kinase C to initiate a variety of downstream cellular processes. DAG kinases attenuate such responses by converting DAG to phosphatidic acid.

This protocol describes an assay designed to quantify cellular DAG levels. The assay exploits the enzymatic conversion of DAG (sn-1,2-diacylglycerol) to phosphatidic acid (1,2-diacyl- sn-glycerol-3-phosphate) in conjunction with the incorporation of a radiolabeled phosphate group by DAG kinase (Figure 1). This assay was described in (Strijbis et ...
[摘要]  二酰基甘油(DAG)是具有不同生物学作用的生物活性脂质。 DAG在接受激活磷脂酶C以切割磷脂的适当刺激时在膜中瞬时累积。 所得水解产物DAG结合蛋白质例如蛋白激酶C以启动各种下游细胞过程。 DAG激酶通过将DAG转化为磷脂酸来减弱这种应答。该方案描述了设计用于定量细胞DAG水平的测定。 该测定利用DAG(1,2-二酰基甘油)到磷脂酸(1,2-二酰基-sn-甘油-3-磷酸)的酶促转化 结合DAG激酶掺入放射性标记的磷酸基团(图1)。 该测定在(Strijbis等人,2013)中描述。