title: Development and characterization of monoclonal antibodies to detect klotho.
abstract: Although antibodies are commercially available to allow investigation into the biology of the age-regulating protein Klotho, problems with antibody specificity and application functionality are significant barriers to progress. Chief among these limitations is the inability of current tools to allow in vivo validation of binding partners originally identified through transfection of tagged proteins. To overcome this barrier, we generated a series of hybridoma cell lines by immunizing rats with a GST-KL1 fusion protein. Purified antibodies generated from these cell lines differentially detect human or mouse Klotho protein via Western blot, immunocyto/histochemistry, and immunoprecipitation. Specificity of antibody binding to Klotho was confirmed by mass spectrometry following immunoprecipitation. With this confidence in antibody specificity, co-immunoprecipitation was utilized to validate the interaction of Klotho/FGFR and Klotho/wnt7a in mouse kidney lysates.
authors.affiliation: 1 Department of Neurobiology, University of Alabama at Birmingham , Alabama.
authors.forename: Astha
authors.initials: A
authors.lastname: Maltare
authors.forename: Angela K
authors.initials: AK
authors.lastname: Nietz
authors.forename: Ann M
authors.initials: AM
authors.lastname: Laszczyk
authors.forename: Taylor S
authors.initials: TS
authors.lastname: Dunn
authors.forename: Mary E
authors.initials: ME
authors.lastname: Ballestas
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Accavitti-Loper
authors.forename: Gwendalyn D
authors.initials: GD
authors.lastname: King
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D005966:Glucuronidase / Q000276:immunology*; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D000090265:Klotho Proteins; D013058:Mass Spectrometry; D051379:Mice; D010957:Plasmids / Q000235:genetics; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D005966:Glucuronidase; D000090265:Klotho Proteins