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

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

Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China
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Authors to whom correspondence should be addressed.
Academic Editor: Julian Borejdo
Received: 3 May 2016 / Revised: 2 June 2016 / Accepted: 8 June 2016 / Published: 17 June 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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. View Full-Text
Keywords: AFAP-120; polyclonal antibody; NINS fragment; AFAP-110; B-cell epitope AFAP-120; polyclonal antibody; NINS fragment; AFAP-110; B-cell epitope
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MDPI and ACS Style

Chen, Y.; Liu, Y.; Guo, J.; Tang, T.; Gao, J.; Huang, T.; Wang, B.; Liu, S. Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD. Int. J. Mol. Sci. 2016, 17, 942.

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