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Mammalian Protein Extraction Reagent (M-PER) supplemented with 50 mM dithiothreitol

M-PER TM哺乳动物蛋白提取试剂

Company: Thermo Fisher Scientific
Catalog#: 78501
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Microsome Isolation from Tissue
[Abstract]  This protocol details the extraction of microsomes from frozen tissue in order to further examine the protein-protein interactions occurring within the endoplasmic reticulum. This protocol was adapted from Abisambra et al. (2013) with modifications made in order to optimize for subsequent use. [摘要]  该协议详细描述了从冷冻组织中提取微粒体,以进一步检查在内质网内发生的蛋白质 - 蛋白质相互作用。 该方案由Abisambra等人 (2013)进行了修改,以优化后续使用。

Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay)
[Abstract]  Autophagy is a dynamic cellular event that is involved in the degradation of long lived proteins and organelles in cells. Biochemical methods such as western blot to measure autophagic proteins have been increasingly used in autophagy studies because it is convenient and objective. Among them, the total amount of Microtubule-associated protein light chain 3 (LC3), the mammalian homologue of the autophagy-related Atg8 in yeast, is a very useful and most commonly used tool in autophagy studies. Other methods such as electron microscopy and immunofluorescence are also available to measure autophagy. The following protocol describes three simple and commonly used protocols for measuring autophagy in cells: LC3B immunofluorescence, western blot and acridine orange assay. Although these three ... [摘要]  自噬是参与细胞中长寿蛋白和细胞器的降解的动态细胞事件。生物化学方法如蛋白质印迹测量自噬蛋白已被越来越多地用于自噬研究,因为它是方便和客观的。其中,微管相关蛋白轻链3(LC3),酵母中自噬相关Atg8的哺乳动物同源物的总量是自噬研究中非常有用和最常用的工具。其他方法如电子显微镜和免疫荧光也可用于测量自噬。以下方案描述了用于测量细胞中自噬的三种简单且常用的方案:LC3B免疫荧光,western印迹和吖啶橙测定。虽然这三种方法经常用于提供关于自噬的基本信息,但是人们应该记住,由于这种动态过程的复杂性,它们不足以给出关于自噬通量的确切细节。此外,吖啶橙测定法仅是检测自噬的补充方法,因为其也对其他细胞器如溶酶体具有高亲和力。对于化合物对自噬通量的影响的进一步研究,推荐另外的和更复杂的测定。这里的协议提供了一个起点,人们得到一个化合物是否可以影响组织培养细胞自噬的快照。

Western Blot for Detecting Phosphorylated STAT3
[Abstract]  The STAT3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors and is constitutively activated in a number of human tumors and possesses oncogenic potential and anti-apoptotic activities. STAT3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding. Western blot is most commonly used to detect the activation of STAT3 by using an antibody that is specific for the phosphorylated tyrosine705. [摘要]  STAT3转录因子是许多细胞因子和生长因子受体的重要信号分子,并且在许多人肿瘤中被组成型激活,并且具有致癌潜力和抗凋亡活性。 STAT3被Tyr705处的磷酸化激活,其诱导二聚化,核转位和DNA结合。 蛋白质印迹最常用于通过使用对磷酸化酪氨酸特异的抗体来检测STAT3的活化。