| Ubiquitination Assay for Mammalian Cells
|
|
Author:
Date:
2016-07-20
[Abstract] Ubiquitin is an 8.5 kDa protein that can be activated and conjugated by ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, respectively. Ubiquitin E3 ligases then recognize protein substrates, and then transfer the ubiquitin from E2 to the targeted protein. This biological process is called ubiquitination, and it is an important biological process which can signal protein degradation via the proteasome. The aim of this protocol is to describe a procedure that determines the level of cellular ubiquitination in a protein-of-interest relative to control cells.
[摘要] 泛素是8.5kDa的蛋白质，其可以分别由泛素激活酶E1和泛素缀合酶E2激活和缀合。 然后泛素E3连接酶识别蛋白质底物，然后将泛素从E2转移到目标蛋白质。 这种生物过程称为泛素化，它是一个重要的生物过程，可以通过蛋白酶体信号蛋白降解。 该方案的目的是描述确定相对于对照细胞的目的蛋白质中的细胞泛素化水平的程序。
|
|
|
| Hepatitis C virus Cell-to-cell Spread Assay
|
|
Author:
Date:
2014-12-20
[Abstract] Hepatitis C virus (HCV) can infect naïve cells via entry of “cell-free” extracellular virus or direct “cell-to-cell” transmission. Here, we describe an assay for detecting HCV cell-to-cell transmission, using a non-growing cell culture system that avoids confounding effects of cell growth. The assay consists of infecting a small number of cells in a confluent monolayer and then blocking subsequent cell-free extracellular virions with a neutralizing antibody such that only cell-to-cell transmission may occur. Under these conditions, incubation at 37 °C results in the formation of infected cell ...
[摘要] 丙型肝炎病毒（HCV）可通过"无细胞"细胞外病毒的进入或直接的"细胞到细胞"传递而感染初始细胞。 在这里，我们描述检测HCV细胞到细胞传播，使用一个非增长的细胞培养系统，避免细胞生长的混杂效应的测定。 该测定法包括感染汇合单层中的少量细胞，然后用中和抗体阻断随后的无细胞的细胞外病毒粒子，使得仅可发生细胞与细胞的传递。 在这些条件下，在37℃下孵育导致感染的细胞病灶的形成。 然后可以通过计数每个焦点中的细胞数量来确定细胞与细胞扩散的程度。 可以修改测定以评估抑制剂和/或特异性细胞基因对HCV的细胞与细胞扩散的影响。
|
|
|