title: Characterization of a rabbit polyclonal antibody against threonine-AMPylation.
abstract: An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.
authors.affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Yi-Heng
authors.initials: YH
authors.lastname: Hao
authors.forename: Trinette
authors.initials: T
authors.lastname: Chuang
authors.forename: Haydn L
authors.initials: HL
authors.lastname: Ball
authors.forename: Phi
authors.initials: P
authors.lastname: Luong
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Ruben D
authors.initials: RD
authors.lastname: Flores-Saaib
authors.forename: Kim
authors.initials: K
authors.lastname: Orth
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007106:Immune Sera; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D000906:Antibodies; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D013912:Threonine; D000249:Adenosine Monophosphate; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein