| Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
|
|
Author:
Date:
2016-10-05
[Abstract] This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of ...
[摘要] 该协议描述了通过激酶PINK1使用重组蛋白的泛素和帕金蛋白的体外磷酸化。两种底物，泛素和帕金蛋白，在保守的丝氨酸65残基（pS65-泛素和pS65-帕金蛋白）上磷酸化。该方案还包括使用单体和K48和K63连接的聚泛素链作为替代底物。虽然有市售的抗体，我们没有测试他们在这个测定中的性能，因为，但使用我们实验室的验证抗体。另外描述了另一种抗体非依赖性方法，使用phos-标记凝胶检测pS65-泛素和pS65-帕金。
[背景] 在细胞中， PINK1是稳定和激活的线粒体膜去极化和其他形式的应力，导致线粒体损伤。活化的PINK1磷酸化泛素，其作为线粒体表面上胞质E3泛素连接酶Parkin的受体。 Parkin对PINK1的磷酸化是Parkin对线粒体底物的完全活性所必需的。活性pS65-Parkin的存在在前馈机制中扩增了作为线粒体标记的线粒体上的pS65-泛素的量。最终，受损的线粒体被自噬噬菌体衔接子识别，并将被蛋白酶体和自噬（mitophagy）降解。这种关键的线粒体质量控制通路促进线粒体的周转，并防止可导致细胞变性的功能障碍线粒体的积累。 PINK1或Parkin中的功能缺失突变与早发性帕金森病相关。
...
|
|
|
| In vitro Detection of S-acylation on Recombinant Proteins via the Biotin-Switch Technique
|
|
Author:
Date:
2014-11-20
[Abstract] Protein palmitoylation is the post-translational modification of proteins via the attachment of palmitate through acyl linkages. The nucleophile sulfhydryl group of cysteines is the common palmitoylation site. Covalent attachment of palmitate occurs on numerous proteins and is usually associated with directing protein localization to the endomembrane system. Detection of protein palmitoylation by in vivo labeling with tritium-labeled palmitic acid typically requires an autoradiographic exposure time of several months, and, thus is not suitable for rapid analyses. Here, we described ...
[摘要] 蛋白质棕榈酰化是通过棕榈酸酯通过酰基键连接的蛋白质的翻译后修饰。半胱氨酸的亲核巯基是常见的棕榈酰化位点。棕榈酸酯的共价附着发生在许多蛋白质上，并且通常与将蛋白质定位到内膜系统相关。通过体内标记氚标记的棕榈酸来检测蛋白质棕榈酰化通常需要几个月的放射自显影曝光时间，因此不适合快速分析。在这里，我们描述了使用拟南芥蛋白激酶（PBS1）作为实例的快速体外检测蛋白S-酰化的简单方案。为了确定PBS1是否通过硫酯键连接到酰基修饰，我们采用"生物素开关"测定法（Hemsley等人，2008）。这项工作首次发表在Qi。et al。（2014），但我们在这里扩展方法。 PBS1在植物的基础免疫系统内起作用，并且是细菌半胱氨酸蛋白酶AvrPphB的靶（Shao等人，2002; Zhang等人，2010） 。它含有预测的N-末端S - 酰基化基序（MGCFSCFDS），其中Cys-3和Cys-6残基预测为被CSS-Palm 3.0棕榈酰化（http：//csspalm.biocuckoo。 org /; Ren等人，2008）。我们的方法利用羟胺诱导的硫酯键裂解，这导致游离的巯基，然后可以与生物素衍生物1-生物素酰氨基-4- [4' - （马来酰亚胺甲基）环己烷甲酰胺基] - 丁烷（生物素-BMCC）缀合。通过蛋白质印迹用链霉亲和素 - ...
|
|
|
| Binding to Secreted Bone Matrix in vitro
|
|
Author:
Date:
2014-02-20
[Abstract] This method examines the bone matrix binding capacity of proteins. Using osteogenic differentiation medium, multipotent stromal cells (MSC) are induced to differentiate into osteocytes in vitro and to secrete bone matrix. The latter is confirmed using Alizarin red S staining, which detects the presence of calcific deposits (hydroxyapatite). These calcific deposits are used to test the bone binding properties of proteins. The binding to the calcific deposits is assessed by Western blot analysis.
[摘要] 这种方法检查蛋白质的骨基质结合能力。 使用成骨分化培养基，诱导多潜能基质细胞（MSC）在体外分化成骨细胞和分泌骨基质。 后者使用茜素红S染色证实，其检测钙化沉积物（羟基磷灰石）的存在。 这些钙沉积物用于测试蛋白质的骨结合性质。 通过蛋白质印迹分析评估与钙化沉积物的结合。
|
|
|