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抗人肌动蛋白丝相关蛋白120kD多克隆抗体的制备与鉴定

Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD.

作者信息

Chen Yujian, Liu Yong, Guo Jiayu, Tang Tao, Gao Jian, Huang Tao, Wang Bin, Liu Shaojun

机构信息

Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China.

出版信息

Int J Mol Sci. 2016 Jun 17;17(6):942. doi: 10.3390/ijms17060942.

DOI:10.3390/ijms17060942
PMID:27322249
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4926475/
Abstract

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.

摘要

肌动蛋白丝相关蛋白120kD（AFAP - 120）是肌动蛋白丝相关蛋白110kD（AFAP - 110）的一种可变剪接异构体，除了与AFAP - 110具有相同结构域外，还包含一个额外的神经元插入（NINS）片段。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，并在神经元分化过程中参与动态肌动蛋白结构的组织。然而，抗AFAP - 120抗体在商业上仍不可得，这可能会阻碍对AFAP - 120功能的研究。在本研究中，我们同时使用ABCpred在线服务器和BepiPred 1.0服务器预测人AFAP - 120蛋白独特的NINS序列中的B细胞表位，发现一个16氨基酸肽序列是这两种工具预测的共有表位。该肽经化学合成并用作免疫原，以制备抗AFAP - 120多克隆抗体（抗AFAP - 120）。通过免疫印迹、免疫沉淀和免疫荧光分析对抗AFAP - 120的敏感性和特异性进行了分析。我们的结果表明，抗AFAP - 120在变性条件下可与过表达的和内源性人AFAP - 120蛋白反应，但不与人AFAP - 110蛋白反应。此外，天然人AFAP - 120蛋白也能被抗AFAP - 120抗体识别。这些结果表明，所制备的抗AFAP - 120抗体将是研究AFAP - 120生化和生物学功能的有用工具。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3c78/4926475/8a454dcac2d5/ijms-17-00942-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3c78/4926475/5ad7973c8ea1/ijms-17-00942-g001.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3c78/4926475/8a454dcac2d5/ijms-17-00942-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3c78/4926475/5ad7973c8ea1/ijms-17-00942-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3c78/4926475/d240ca70a860/ijms-17-00942-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3c78/4926475/0f9cf68b7a74/ijms-17-00942-g003.jpg
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The actin cross-linking protein AFAP120 regulates axon elongation in a tyrosine phosphorylation-dependent manner.
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