Preparation and Characterization of a Polyclonal Antibody against Human Actin Filament-Associated Protein-120 kD.
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.
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. triser@126.com.
Yujian
Y
Chen
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. liuyongxiao1225@hotmail.com.
Yong
Y
Liu
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. gjy992@outlook.com.
Jiayu
J
Guo
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. taotang006@163.com.
Tao
T
Tang
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. gaojian1918@163.com.
Jian
J
Gao
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. huang_tao945@163.com.
Tao
T
Huang
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. xiechuanbin2003@163.com.
Bin
B
Wang
Department of Neurobiology, Institute of Basic Medical Sciences, Beijing 100850, China. liusj@bmi.ac.cn.
Shaojun
S
Liu
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000915:Antibody Affinity; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D000939:Epitopes / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
C084510:AFAP 110; D000906:Antibodies; D000939:Epitopes; D008840:Microfilament Proteins; D010750:Phosphoproteins
AFAP-110; AFAP-120; B-cell epitope; NINS fragment; polyclonal antibody