title: Generation of Domain-Specific Monoclonal Antibodies Against Human Glutaredoxin3.
abstract: Human Glutaredoxin3 (hGLRX3), which encodes a 37.4 kDa protein, possesses an N-terminal Trx homology domain followed by two tandem repeats of Grx domains. GLRX3 is expressed in many tissues and plays important roles in iron metabolism, antioxidant effect, cell proliferation and development, regulation of immune reaction, and tumorigenesis. The mechanisms underlying the biological function of GLRX3 are still not clear. To facilitate the functional research of GLRX3, in this study, monoclonal antibodies (MAbs) against hGLRX3 were produced by using purified prokaryotic recombinant 6His-hGLRX3 fusion protein as the immunogen. Five MAbs were obtained after preliminary screening by indirect enzyme-linked immunosorbent assay, then further characterized by Western blot analysis and immunocytochemistry. The domain specificity of these MAbs was also evaluated. Owing to the high conservation of protein sequences among different species, anti-GLRX3 MAbs produced in this study were shown to be immunoactive for GLRX3 in the cells from other species, such as mice, rats, Chinese hamster, and zebrafish. These domain-specific anti-GLRX3 MAbs will be an essential tool to investigate the roles of GLRX3 in normal physiological or pathological conditions.
authors.affiliation: 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China .
authors.forename: Xin
authors.initials: X
authors.lastname: Dai
authors.affiliation: 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China .
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Li
authors.affiliation: 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China .
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Sun
authors.affiliation: 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China .
authors.forename: Kai
authors.initials: K
authors.lastname: Cai
authors.affiliation: 2 Department of Pathology, Northwestern University Feinberg School of Medicine , Chicago, Illinois.
authors.forename: Qinwen
authors.initials: Q
authors.lastname: Mao
authors.affiliation: 1 Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University , Xi'an, China .
authors.forename: Haibin
authors.initials: H
authors.lastname: Xia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D000918:Antibody Specificity; D015153:Blotting, Western; D016466:CHO Cells; D002352:Carrier Proteins / Q000008:administration & dosage / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000032:analysis / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013154:Spleen / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D015027:Zebrafish
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C404105:GLRX3 protein, human; D011993:Recombinant Fusion Proteins
keywords: Glutaredoxin3; hybridoma; monoclonal antibody; prokaryotic expression