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Methanol anhydrous, 99.8%

甲醇

Company: Sigma-Aldrich
Catalog#: 322415
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Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells
Author:
Date:
2018-04-20
[Abstract]  Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. The aggregative adherence fimbria II (AAF/II) is the most important adherence factor of EAEC prototype strain 042 (EAEC042) to intestinal cells. Multiple receptors for AAF/II on epithelial cells have been identified including the transmembrane signaling mucin Muc1. This protocol describes a method to measure adherence of EAEC strains to HEK293 cells expressing the Muc1 glycoprotein. [摘要]  肠道集聚性大肠杆菌(EAEC)是全球儿童和成人急性腹泻的公认原因。 EAEC菌株的特征在于存在聚集粘附菌毛(AAF),其通过介导与肠粘膜的附着和通过引发宿主炎症反应而在发病机制中起关键作用。 聚合粘附菌毛II(AAF / II)是EAEC原型菌株042(EAEC042)对肠细胞最重要的粘附因子。 已经鉴定了上皮细胞上AAF / II的多种受体,包括跨膜信号传导粘蛋白Muc1。 该协议描述了测量EAEC菌株对表达Muc1糖蛋白的HEK293细胞的依从性的方法。

【背景】EAEC是世界范围内地方性和流行性腹泻病的重要原因。尽管发展中国家儿童腹泻最常见,但EAEC还与免疫受损成人腹泻,旅行者和工业化国家的食源性疾病有关,例如由志贺毒素(Stx)2a型产生的大致致命爆发2011年在北欧的血清型O104:H4的EAEC菌株(Harrington等人,2006; Rasko等人,2011)。 EAEC发病机制由生物体粘附肠细胞,产生肠毒素和细胞毒素并最终诱导炎症的能力决定(Harrington等,2006)。 EAEC对肠细胞的依从性由AAF菌毛粘附素介导(Czeczulin等人,1997)。迄今为止,已经描述了至少5种AAF菌毛的变体,全部编码在范围为55至65MDa的毒力质粒中(Jonsson等人,2015)。 ...

Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
Author:
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, ...

MPM-2 Mediated Immunoprecipitation of Proteins Undergoing Proline-directed Phosphorylation
Author:
Date:
2016-12-05
[Abstract]  Immunoprecipitation (IP) represents a widely utilized biochemical method to isolate a specific protein from a complex mixture taking advantage of an antibody that specifically recognizes that particular target molecule. This procedure is extremely versatile and can be applied to concentrate a specific protein, to identify interacting partners in complex with it or to detect post-translational modifications. The mitotic protein monoclonal 2 (MPM-2) is an antibody originally raised against extracts of synchronized mitotic HeLa cells to identify proteins selectively present in mitotic, and not in interphase-cells (Davis et al., 1983). MPM-2 recognizes phosphorylated serine or threonine residues followed by proline (pS/T-P), consensus epitopes generated by the concerted action of ... [摘要]  免疫沉淀(IP)代表广泛使用的生物化学方法,以利用特异性识别特定靶分子的抗体从复杂混合物中分离特异性蛋白质。该程序是非常通用的,可以应用于集中特定蛋白质,识别与其复合的相互作用的配偶体或检测翻译后修饰。有丝分裂蛋白单克隆2(MPM-2)是最初针对同步有丝分裂HeLa细胞的提取物产生的抗体,以鉴定选择性存在于有丝分裂中的蛋白质,而不是在间期细胞中(Davis等,1983)。 MPM-2识别磷酸化的丝氨酸或苏氨酸残基,随后是脯氨酸(pS / T-P),由脯氨酸指导的蛋白激酶和磷酸酶的共同作用产生的共有表位(Lu等,2002)。这些可逆磷酸化事件已经出现以通过促进目标上的构象变化来控制各种细胞过程,这不仅仅是由于磷酸化事件本身。这些基序一旦被磷酸化,就能够招募Pin1(肽基 - 脯氨酰异构酶NIMA相互作用蛋白1)(Lu等人,1996; Lu和Zhou,2007),这是一个促进肽键上的顺式/反式异构化反应的伴侣,在基础功能不同的构象之间切换底层(Lu,2004; Wulf等人,2005)。该方案描述了使用支架分子突触后密度蛋白-95(PSD-95)(Chen等人,2005),神经元Pin1靶(Antonelli等,2016)作为示例的基于MPM-2的免疫沉淀策略以说明详细的程序。
【背景】由MPM-2抗体识别的抗原的鉴定代表了发现经过磷酸化脯氨酰异构化调控机制的靶分子的有用起点。 ...

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