C14orf119的表达纯化及其多克隆抗体的制备。

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2025-09-22 DOI:10.1016/j.pep.2025.106822

Yong-Hong Nie , Qiang Li , Yan-Ji Lu , Tuo Tang , Xian Hong , Xin-Xin Yue , Zhi-Hui Deng , Tao Wang

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引用次数: 0

摘要

C14orf119是一种功能未知的线粒体蛋白，可能与缺血性卒中发病机制有关。为了进行全面的生物学研究，我们开发并验证了针对该靶点的特异性多克隆抗体。我们的方法包括在大肠杆菌中对His-和gst标记的C14orf119进行原核表达和层析纯化。纯化后的His-C14orf119作为兔多克隆抗体生产的免疫原，纯化后的GST-C14orf119用于随后的目标特异性抗体亲和纯化。严格的鉴定表明该抗体对外源性和内源性C14orf119都具有特异性。值得注意的是，C14orf119抗体未能检测到对照his标记的蛋白，证实了在基于gst的亲和纯化过程中有效去除了抗his抗体。这种经过严格验证的抗体为C14orf119的功能表征提供了关键的分子工具，为其病理生理作用的机制研究铺平了道路。

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Expression and purification of C14orf119 and generation of its polyclonal antibody

C14orf119 is a functionally uncharacterized mitochondrial protein potentially implicated in ischemic stroke pathogenesis. To enable comprehensive biological studies, we developed and validated a specific polyclonal antibody against this target. Our approach involved prokaryotic expression and chromatographic purification of both His- and GST-tagged C14orf119 in Escherichia coli. The purified His-C14orf119 served as immunogen for rabbit polyclonal antibody production, while the purified GST-C14orf119 enabled subsequent affinity purification of target-specific antibody. Rigorous characterization demonstrated the antibody's exclusive specificity for both exogenous and endogenous C14orf119. Notably, the C14orf119 antibody failed to detect control His-tagged proteins, confirming effective removal of anti-His antibodies during the GST-based affinity purification process. This rigorously validated antibody provides a critical molecular tool for functional characterization of C14orf119, paving the way for mechanistic investigations into its pathophysiological roles.

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.