| Cell Surface Protein Detection to Assess Receptor Internalization
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Author:
Date:
2016-10-20
[Abstract] The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. ...
[摘要] 膜受体在暴露于不同的刺激剂/抑制剂时的迁移是非常重要的。其中，膜受体的内化影响其对配体的可及性和对环境线索的细胞应答性。在实验上，受体内化可用作其活化的量度。在我们的研究中，我们采用这种方法来探讨神经肽Y（NPY）的七个跨膜结构域受体，Y5R和脑源性神经营养因子（BDNF），TrkB的酪氨酸激酶受体之间的串扰。为此，我们测量了用TrkB配体BDNF刺激后Y5R的内化。在用BDNF处理后，将细胞暴露于选择性标记表面蛋白的不透膜的生物素化试剂。随后，生物素化的膜蛋白在具有抗生物素蛋白树脂的柱上亲和纯化，并通过蛋白质印迹分析。存在于对照和配体处理的细胞的细胞表面上的受体部分的差异用作其内化和对特定刺激的反应的量度。
[Backg 回合] 可以使用两种主要策略 - 显微镜和生物化学来测量响应外部刺激的细胞膜受体内化。最常见的方法是使用显微镜 - ...
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| Detection of Wnt5 in Media Conditioned by Mouse Embryonic Fibroblast
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Author:
Date:
2016-10-20
[Abstract] This protocol describes the procedure of visualizing secreted Wnt5 protein in serum free media via western blotting. This procedure can also be used to visualize other secreted proteins larger than 10,000 daltons. The work presented in this paper visualizes Wnt5 secreted by mouse embryonic fibroblast (MEF), but can be adapted to other cell lines including those transiently transfected by plasmids.
[摘要] 该协议描述了通过免疫印迹观察无血清培养基中分泌的Wnt5蛋白的过程。 该程序还可用于显现大于10,000道尔顿的其他分泌的蛋白质。 本文提出的工作可视化由小鼠胚胎成纤维细胞（MEF）分泌的Wnt5，但可以适应于其他细胞系，包括由质粒瞬时转染的那些。
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| Displacement-based ELISA: Quantifying Competition between Two Binding Partners for Interaction with a His-tagged Ligand Immobilized on a Ni2+-NTA Plate
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Author:
Date:
2016-03-05
[Abstract] The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant conditions (i.e. in the presence of Zn2+ or with EDTA to chelate Zn2+) and then allowed to compete for binding to S6 which was maintained in limiting concentration. The result was obtained through an ELISA, where S6-His is first bound to a Ni2+-NTA plate, followed by addition of S18-2 in excess to S6, then by addition of increasing concentrations of S18-1. The percentage of S18-2 that remained bound to S6 was ...
[摘要] 设计置换测定以定量来自结核分枝杆菌（Mycobacterium tuberculosis）S18-1和S18-2的两个同源核糖体蛋白质之间的直接竞争，用于与它们的同源结合伴侣，核糖体蛋白S6（Prisic et al。，2015）。 S18蛋白质在两种生理相关条件下（即在Zn 2+ 2+存在下或用EDTA螯合Zn 2+ 2+）进行透析，并且然后允许竞争结合S6，其维持在有限浓度。通过ELISA获得结果，其中S6-His首先结合Ni 2+ 2+ -NTA板，然后加入超过S6的S18-2，然后加入递增浓度的S18-1。在化学发光ELISA中用S18-2蛋白和第二抗体特异性的抗体定量保留结合S6的S18-2的百分比。以这种方式，S18-1蛋白质被S18-1蛋白质的置换报告为通过用S18-2饱和S6实现的全强度信号的百分比。在其基础上，该方法利用天然蛋白质 - 蛋白质相互作用，并且可以应用于其中两种或更多种蛋白质竞争结合上述靶配体的其他系统。
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