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.
Copyright © 2010 Elsevier B.V. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Monophosphate / chemistry
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Adenosine Monophosphate / immunology*
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Adenosine Monophosphate / metabolism
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Animals
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Antibodies / chemistry*
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Antibodies / immunology
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Antibodies / metabolism
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Blotting, Western
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Cloning, Molecular
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GTP Phosphohydrolases / chemistry
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GTP Phosphohydrolases / metabolism
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HeLa Cells
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Humans
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Immune Sera
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Protein Processing, Post-Translational
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Rabbits
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Threonine / chemistry
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Threonine / immunology*
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Threonine / metabolism
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cdc42 GTP-Binding Protein / chemistry
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cdc42 GTP-Binding Protein / immunology
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cdc42 GTP-Binding Protein / metabolism
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rac1 GTP-Binding Protein / chemistry
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rac1 GTP-Binding Protein / immunology
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rac1 GTP-Binding Protein / metabolism
Substances
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Antibodies
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Immune Sera
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Recombinant Fusion Proteins
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Threonine
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Adenosine Monophosphate
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GTP Phosphohydrolases
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cdc42 GTP-Binding Protein
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rac1 GTP-Binding Protein