无标记协议，使用等温滴定量热法和生物层干涉法对人 eIF5 模拟蛋白进行蛋白质亲和力定量。
Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein.
机构信息
Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA.
出版信息
STAR Protoc. 2022 Aug 16;3(3):101615. doi: 10.1016/j.xpro.2022.101615. eCollection 2022 Sep 16.
eIF5-mimic protein (5MP) controls translation through its interaction with eukaryotic translation initiation factor (eIF) 2 and eIF3 and alters non-AUG translation rates for oncogenes in cancer and repeat expansions in neurodegenerative disease. To precisely evaluate the effect of 5MP mutations on binding affinity against eIFs, here we describe two label-free protocols of affinity measurement for 5MP binding to eIF2 or eIF3 protein segments, termed isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI), starting with how to purify proteins used. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
eIF5 模拟蛋白 (5MP) 通过与真核翻译起始因子 (eIF) 2 和 eIF3 的相互作用来控制翻译，并改变癌症中的癌基因和神经退行性疾病中的重复扩展的非 AUG 翻译速率。为了精确评估 5MP 突变对与 eIFs 结合亲和力的影响，我们在这里描述了两种针对 5MP 与 eIF2 或 eIF3 蛋白片段结合的无标记亲和力测量方案，即等温滴定量热法 (ITC) 和生物层干涉测量法 (BLI)，首先介绍如何纯化所用的蛋白质。有关此协议的使用和执行的完整详细信息，请参阅 Singh 等人。(2021)。
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