基于 HABA 染料的比色测定法,用于检测含阿维丁融合蛋白中未被占用的生物素结合位点。

IF 2.2 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS BioTechniques Pub Date : 2024-09-13 DOI:10.1080/07366205.2024.2397288
Sonia Mukherjee,Pierre Leblanc,Mark C Poznansky,Ann E Sluder
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引用次数: 0

摘要

阿维丁-生物素结合是自然界中最强大的非共价蛋白质-配体相互作用,在生物技术领域有着广泛的应用。这些应用中经常遇到的一个难题是如何准确确定含阿维丁融合蛋白中未被占用的生物素结合位点的数量。我们根据阴离子染料 4'-hydroxyazobenzene-2-carboxylic acid 与阿维丁内生物素结合位点的亲和力,制定了一种微型化的新型检测方案,以量化可用的生物素结合位点。我们将这种检测方法用作质量控制检测,以评估不同融合蛋白生产批次中可用生物素结合位点的数量。与常用的基于荧光的检测方法相比,这种方法可简化生物素结合的评估,比其他方法耗时更少,而且适用于各种融合蛋白。
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A HABA dye-based colorimetric assay to detect unoccupied biotin binding sites in an avidin-containing fusion protein.
Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4'-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.
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来源期刊
BioTechniques
BioTechniques 工程技术-生化研究方法
CiteScore
4.40
自引率
0.00%
发文量
68
审稿时长
3.3 months
期刊介绍: BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies. Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers: Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices Surveys of technical approaches related to broad fields of research Reviews discussing advancements in techniques and methods related to broad fields of research Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
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