| Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay)
|
|
Author:
Date:
2013-12-20
[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 ...
[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。
|
|
|
| Cross-linked RNA Immunoprecipitation
|
|
Author:
Date:
2013-03-05
[Abstract] This method is for the immunoprecipitation of Flag-Tagged RNA binding proteins from mammalian cell lines and isolation of the bound RNAs for analysis by quantitative real-time PCR. The RNA binding protein of interest should be tagged with the M2 Flag-tag and expressed in the mammalian cell line of interest (Knuckles et al., 2012). However, specific antibodies for the protein of interest can be used in conjunction with Sepharose G-beads.
[摘要] 该方法用于来自哺乳动物细胞系的Flag-标记的RNA结合蛋白的免疫沉淀和分离结合的RNA，用于通过定量实时PCR分析。 感兴趣的RNA结合蛋白应该用M2 Flag标签标记并在感兴趣的哺乳动物细胞系中表达（Knuckles等人，2012）。 然而，感兴趣的蛋白质的特异性抗体可以与Sepharose G-珠结合使用。
|
|
|