相关实验视频

Updated: May 6, 2026

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Published on: September 28, 2018

-SrcKD的协议:rPTPεD1复合制剂和BLI结合试验,以证明它们的外位界面

Nadendla EswarKumar¹, Kumar TewarySunil², Meng-Chiao Ho³

¹Institute of Biological Chemistry, Academia Sinica, 128 Academia Road Sec. 2, Nankang, Taipei 115, Taiwan; Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

|July 3, 2024

概括

本研究详细介绍了 Src 激酶域 (SrcKD) 和其与 rPTPεD1.1 复合物的体外酸化协议. 这些方法允许使用生物层干涉测量 (BLI) 来确定结合亲和力.

科学领域:

生物化学生物化学
分子生物学分子生物学
结构生物学结构生物学

背景情况:

Src 激酶域 (SrcKD) 是细胞信号通路中的一个关键调节器.
蛋白氨酸酸酶 (PTPs) 和rPTP epsilon (rPTPε) 一样,在去酸化基质中发挥关键作用,例如Src.
了解化SrcKD和PTP域之间的相互作用对于破译信号机制至关重要.

研究的目的:

为 Src 激酶域 (SrcKD) 的体外酸化建立一个全面的协议.
为了描述光-SrcKD:rPTPεD1复合物的制备,用于生物物理特征.
概述一种基于生物层干扰测量 (BLI) 的结合试验,用于量化-SrcKD与rPTPεD1.0之间的相互作用.

主要方法:

在实验室中使用纯化酶对复合SrcKD进行酸化.
在化SrcKD和rPTPepsilon (rPTPεD1) 的D1域之间形成复合体.
生物层干扰计 (BLI) 用于实时结合动力学和亲和度测量.
微角X射线散射 (SAXS) 用于对该综合体的结构洞察 (被提及为准备步骤).

主要成果:

为生成化SrcKD建立了一个强大的协议.
基-SrcKD:rPTPεD1复合体已经成功准备好.
BLI测定提供了关于-SrcKD和rPTPεD1.1之间的结合亲和性的定量数据.

结论:

该协议促进了对Src酶酸化及其通过PTPs调节的研究.
开发的方法使得可详细的生物化学和生物物理特征的-SrcKD:rPTPεD1相互作用.
这项工作为进一步研究Src-PTP信号网络提供了基础.

关键词:

蛋白质表达和净化过程信号传导信号传导结构生物学结构生物学

更多相关视频

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Published on: June 30, 2019

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes

Published on: January 12, 2024

相关概念视频

Assembly of Signaling Complexes

Assembly of Signaling Complexes

Multiprotein signaling complexes are formed in a dynamic process involving protein-protein interactions at the cytoplasmic domain of transmembrane receptors or enzymatic and non-enzymatic proteins associated with the receptor. These complexes ensure the activation and propagation of intracellular signals that regulate cell functions.
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...

Receptor Tyrosine Kinases

Receptor Tyrosine Kinases

Receptor tyrosine kinases or RTKs are membrane-bound receptors that phosphorylate specific tyrosine on protein substrates. RTKs regulate cellular growth, differentiation, survival, and migration. They contain an extracellular ligand binding domain, a transmembrane domain, and a cytosolic tail with intrinsic kinase activity. Several extracellular signaling molecules activate RTKs in one or more ways and relay the signal downstream. Ligands such as platelet-derived growth factor (PDGF) or...