Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD.
Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD.
复制标题
抗人肌动蛋白丝相关蛋白 120 kD 多克隆抗体的制备和表征
DOI：
10.3390/ijms17060942
复制
发表时间：
2016-06-17
影响因子：
5.6
通讯作者：
Liu S
中科院分区：
文献类型：
--
作者：
Chen Y;Liu Y;Guo J;Tang T;Gao J;Huang T;Wang B;Liu S
肌动蛋白诱导相关蛋白-120kD（AFAP-120）是肌动蛋白诱导相关蛋白-110kD（AFAP-110）的一种选择性剪接异构体，除了与AFAP-110相同的结构域外，还含有一个额外的神经元插入片段（NINS）。与在组织中广泛表达的AFAP-110不同，AFAP-120在神经系统中特异性表达，并且在神经元分化期间在组织动态肌动蛋白结构中起作用。然而，抗AFAP-120的抗体还没有商业化，这可能会阻碍AFAP-120的功能研究。在本研究中，我们同时使用ABCpred在线服务器和BepiPred 1.0服务器来预测人AFAP-120蛋白的专有NINS序列中的B细胞表位，并且发现16 aa肽序列是两种工具预测的共有表位。化学合成该肽并用作免疫原以制备抗AFAP-120的多克隆抗体（anti-AFAP-120）。用免疫印迹、免疫沉淀和免疫荧光法分析抗AFAP-120的敏感性和特异性。结果表明，抗AFAP-120抗体在变性条件下能与过量表达的内源性人AFAP-120蛋白发生反应，而与人AFAP-110蛋白不发生反应。此外，天然人AFAP-120蛋白也可以被抗AFAP-120抗体识别。结果表明，所制备的抗AFAP-120抗体为进一步研究AFAP-120的生物学功能奠定了基础。
Actin filament-associated protein-120kD (AFAP-120) is an alternatively spliced isoform of actin filament-associated protein-110kD (AFAP-110) and contains an additional neuronal insert (NINS) fragment in addition to identical domains to the AFAP-110. Unlike AFAP-110 widely expressed in tissues, AFAP-120 is specifically expressed in the nervous system and plays a role in organizing dynamic actin structures during neuronal differentiation. However, anti-AFAP-120 antibody is still commercially unavailable, and this may hinder the function research for AFAP-120. In this study, we simultaneously used the ABCpred online server and the BepiPred 1.0 server to predict B-cell epitopes in the exclusive NINS sequence of human AFAP-120 protein, and found that a 16aa-peptide sequence was the consensus epitope predicted by both tools. This peptide was chemically synthesized and used as an immunogen to develop polyclonal antibody against AFAP-120 (anti-AFAP-120). The sensitivity and specificity of anti-AFAP-120 were analyzed with immunoblotting, immunoprecipitation, and immunofluorescence assays. Our results indicated that anti-AFAP-120 could react with over-expressed and endogenous human AFAP-120 protein under denatured condition, but not with human AFAP-110 protein. Moreover, native human AFAP-120 protein could also be recognized by the anti-AFAP-120 antibody. These results suggested that the prepared anit-AFAP-120 antibody would be a useful tool for studying the biochemical and biological functions of AFAP-120.
影响因子：
2.5
作者：
Harder J;Xu X;Letourneau P;Lanier LM
通讯作者：
Lanier LM