title: Production and characterization of polyclonal and monoclonal Abs against the RNA-binding protein QKI.
abstract: RNA-binding protein QKI, a member of the Signal Transduction and Activation of RNA family, is found to be essential in the blood vessel development and postnatal myelination in central nervous system (Woo et al., Oncogene 28:1176-1186, 2009; Lu et al., Nucleic Acids Res 31(15):4616-4624, 2003; Bohnsack et al., Genesis 44(2):93-104, 2006). However, its wide expression pattern suggests other fundamental roles in vivo (Kondo et al., Mamm Genome 10(7):662-669, 1999). To facilitate the understanding of QKI function in various systems, we prepared the polyclonal and monoclonal antibodies against QKI. To obtain the antigen, recombinant His-tagged QKI was expressed in Escherichia coli and highly purified by Ni(2+)-chelated column combined with hydrophobic and ion exchange methods. Following three types of immunizations with different adjuvants, including Freund's, PAGE gel, and nitrocellulose membrane, only the antiserum produced with Freund's adjuvant is effective for Western blot detection. Several McAb clones are able to recognize both endogenous and over-expressed QKI with high affinity in Western blot and immunofluorescence. The specificity of Ab was validated as weakening, and no specific signals were observed in cells with QKI knocking down. Immunohistochemistry analysis further showed positive staining of QKI in kidney where QKI mRNA was abundantly expressed, ensuring the wide applications of the QKI Abs in the ongoing mechanistic studies.
authors.affiliation: Department of Biochemistry and Molecular Biology, the State Key Laboratory of Cancer Biology, The Fourth Military Medical University, Xi'an, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Bo
authors.initials: B
authors.lastname: Huang
authors.forename: Fang
authors.initials: F
authors.lastname: Yu
authors.forename: Mengying
authors.initials: M
authors.lastname: Wei
authors.forename: Guodong
authors.initials: G
authors.lastname: Yang
authors.forename: Haiyan
authors.initials: H
authors.lastname: Fu
authors.forename: Liang
authors.initials: L
authors.lastname: Jin
authors.forename: Liyuan
authors.initials: L
authors.lastname: Bai
authors.forename: Xianli
authors.initials: X
authors.lastname: He
authors.forename: Zifan
authors.initials: Z
authors.lastname: Lu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016601:RNA-Binding Proteins / Q000276:immunology*; D011817:Rabbits
chemical_list: D000911:Antibodies, Monoclonal; C490832:QKI protein, human; D016601:RNA-Binding Proteins