Protein blot assays specific for the discrimination of the centromere autoantigen, CENP-A, from human cells.
Protein blot assays specific for the discrimination of the centromere autoantigen, CENP-A, from human cells.
复制标题
专门用于区分人类细胞中着丝粒自身抗原 CENP-A 的蛋白质印迹测定。
DOI：
10.1002/elps.11501401145
复制
发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Hoch,SO
中科院分区：
文献类型：
--
作者：
Billings,PB;Martinez,A;Haselby,JA;Hoch,SO
蛋白质印迹已被证明是检测与多种自身免疫性疾病相关的离散的、免疫反应性抗原靶点的宝贵资源。随着自身抗原名册的扩大，针对特定多肽抗原定制特定凝胶或印迹条件变得越来越普遍。这里报道了两种这样的检测方法，应用于人类着丝粒蛋白（CENP-A）的分离和可视化，CENP-A是一种与风湿病、系统性硬化症相关的着丝粒自身抗原。着丝粒抗原在 1MMgCl2 存在下有效溶解，以便进一步纯化。 CENP-A 与组蛋白（主要是 H3 和 H4）共纯化。两种 CENP-A 特异性蛋白印迹测定将 CENP-A 与组蛋白分开，并增强 CENP-A 免疫反应性。第一个测定基于使用酸性尿素凝胶，选择 Triton X-100 浓度以最大限度地分离 CENP-A 与所有组蛋白。第二种测定基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，以区分两种分子量相似的非常碱性的蛋白质，即 CENP-A 和组蛋白 H3。对于每个凝胶系统，选择性选择相关的免疫印迹参数可以重复区分 CENP-A 抗原。
The Western or protein blot has proven to be a valuable resource in detecting discrete, immunoreactive antigen targets associated with a variety of autoimmune diseases. As the roster of autoantigens has expanded, it has become increasingly common to tailor specific gel or blot conditions to a particular polypeptide antigen. Two such assays are reported here as applied to the fractionation and visualization of human centromere protein (CENP‐A), a centromere autoantigen associated with the rheumatic disease, systemic sclerosis. The centromere antigens are effectively solubilized in the presence of 1MMgCl2to allow for further purification. CENP‐A copurifies with the histone proteins, primarily H3 and H4. The two CENP‐A‐specific protein blot assays separate CENP‐A from the histone proteins and enhance CENP‐A immuno‐reactivity. The first assay is based on the use of acid‐urea gels with a Triton X‐100 concentration chosen to maximize separation of CENP‐A from all the histones. The second assay is based on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis to differentiate two very basic proteins of similar molecular weight, namely CENP‐A and histone H3. For each gel system, a selective choice of associated immunoblot parameters allows for the reproducible discrimination of the CENP‐A antigen.