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Amplify Fluorographic Reagent

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Company: Cytiva
Catalog#: NAMP100
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Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
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Date:
2017-11-05
[Abstract]  Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to monitor Cathepsin D processing through secretory pathway and secretion using immunoprecipitation, SDS-PAGE and fluorography. [摘要]  通过研究其配体组织蛋白酶D的加工和分泌的改变,可以在活细胞中研究甘露糖-6-磷酸受体功能。在此描述的测定在文献中已经很好地确定,并且包括新合成的蛋白质的代谢标记,其中[35 [S]甲硫氨酸半胱氨酸,以监测组织蛋白酶D处理,通过分泌途径和分泌使用免疫沉淀,SDS-PAGE和荧光。

【背景】组织蛋白酶d(CATD)是一种溶酶体天冬氨酸蛋白酶由甘露糖-6-磷酸受体(M6PRs)排序在哺乳动物细胞中该传输它从反面高尔基体网络到内涵体/溶酶体(戈什等人,2003 )。将CatD合成为前体蛋白(〜52kDa),其在溶酶体中被切割以产生中间体(〜48kDa)或成熟的溶酶体形式(〜34kDa)。微量的前体蛋白质也从生物合成途径分泌(Benes et al。,2008)。 catD的丰度可以使用几种方法来确定,例如基于免疫荧光的染色(Poole等人,1972),蛋白质印迹,荧光活性测定(Bewley等人, (Hirst等人,2009; Kametaka等人,2007; Tavares等人,2011)或用脉冲追踪分析进行代谢标记(Hirst等人,2009; Kametaka等人, ...

Pulse Chase of Suspension Cells
Author:
Date:
2014-07-05
[Abstract]  Pulse-chase method is a powerful technique used to follow the dynamics of proteins over a period of time. The expression level, processing, transport, secretion or half-life of proteins can be tracked by metabolically labeling the cells, such as with radiolabeled amino acids (pulse step). This protocol describes the condition used to study the folding and disulfide bond formation of immunoglobulin in suspension cells. With some minor modifications, this protocol can be adapted to study the degradation rate or the secretion of target proteins. [摘要]  脉冲追踪法是用于跟踪蛋白质在一段时间内的动力学的有力技术。 蛋白质的表达水平,加工,转运,分泌或半衰期可以通过代谢标记细胞,例如用放射性标记的氨基酸(脉冲步骤)来追踪。 该方案描述了用于研究悬浮细胞中免疫球蛋白的折叠和二硫键形成的条件。 通过一些微小的修改,这个协议可以适应于研究目标蛋白的降解速率或分泌。

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