| Measurement of Protein-Protein Interactions through Microscale Thermophoresis (MST)
|
|
Author:
Date:
2020-04-05
[Abstract] The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations resulted in controversy regarding the values of the dissociation constant (Kd) (Freeman et al., 2000). MST is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. The technique is based on the movement of molecules along microscopic temperature gradients, and it detects changes in their hydration shell, charge or size. One binding partner is fluorescently labeled, while the other binding partner remains label-free. We used a protocol that allows the determination of the binding affinity by MST without purification of ...
[摘要] [摘要 ] 近年来，通过表面等离振子共振（SPR）和等温滴定热法（ITC）研究了PD-1和PD-L1的结合相互作用，但这些研究引起了解离常数值的争议。 （ķ d ）（弗里曼等人，2000） 。MST是一种功能强大的新方法，可定量分析低样品消耗的蛋白质-蛋白质相互作用（PPI）。该技术基于分子沿微观温度梯度的移动，并检测其水合壳，电荷或大小的变化。一个结合蛋白摹伴侣荧光拉贝领导， 而另一个绑定伙伴仍保持无标签状态。我们使用的协议允许通过MST确定结合亲和力，而无需从细胞裂解物中纯化靶蛋白。将此MST方法应用于在CHO-K1细胞中表达的PD-1-eGFP和PD-L1-eGFP的应用，首次使我们能够确定PD-1及其配体PD-L1之间形成的复合物的亲和力在肿瘤逃逸期间。该协议在研究蛋白质与小分子之间的相互作用方面具有多种潜在应用。
[背景技术 [ 0002 ] 鉴定能够调节PD-1和PD-L1之间形成的复合物的亲和力的化合物代表了针对肿瘤免疫逃逸的新疗法的开发的重大进展。该方案涉及eGFP 融合蛋白的过表达，然后从细胞裂解物中提取eGFP 融合蛋白，无需任何纯化步骤即可测定PD-1和PD-L1之间的亲和常数。因此，该协议的发展需要产生eGFP 融合蛋白。该协议旨在通过避免繁琐的纯化步骤来定量加速蛋白质相互作用的表征。该协议还可以用于通过PD-1 / ...
|
|
|
| Mitochondrial Biogenesis Assay after 5-day Treatment in PC-3 Cells
|
|
Author:
Date:
2015-01-20
[Abstract] Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial protein synthesis, or mitochondrial biogenesis, relative to the cellular protein synthesis, provides important information on potential mitochondrial toxicity.
[摘要] 药物诱导的线粒体损伤可以由许多不同的机制引起，包括线粒体DNA复制，转录，翻译和改变的蛋白质功能的抑制。 相对于细胞蛋白质合成的线粒体蛋白质合成水平或线粒体生物发生的确定提供了关于潜在线粒体毒性的重要信息。
|
|
|