A monoclonal antibody to the phosphorylated form of glial fibrillary acidic protein: application to a non-radioactive method for measuring protein kinase activities.
Monoclonal antibody YC10 showed specificity for the phosphorylated form of human, bovine and porcine glial fibrillary acidic proteins (GFAPs) and negligible reactivity towards the dephosphorylated form of the GFAPs. Analysis of species specificity and of the epitope, determined using synthetic phosphopeptides, indicated that this antibody recognized the local phosphorylation-site sequence Thr-phosphoSer-Ala-Ala-Arg-Arg (residues 7-12 of GFAP). Making use of this antibody we developed a non-radioactive method to measure protein kinase activities. After incubation of a protein kinase with non-radioactive ATP in ninety-six wells coated with the synthetic peptide Arg-Arg-Arg-Val-Thr-Ser-Ala-Ala-Arg-Arg-Ser-Cys (residues 3-13 of GFAP), the phosphorylated product was detected by using this mouse antibody and peroxidase-labeled goat anti-mouse IgG. This method proved to be equally as sensitive as the radioactive method for the measurement of protein kinase activities and was less affected by concentrations of ATP present in the reaction mixture.
Yano
T
T
Laboratory of Experimental Radiology, Aichi Cancer Center Research Institute, Nagoya, Japan.
Taura
C
C
Shibata
M
M
Hirono
Y
Y
Ando
S
S
Kusubata
M
M
Takahashi
T
T
Inagaki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000201:enzymology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D005904:Glial Fibrillary Acidic Protein; D010748:Phosphopeptides; D011494:Protein Kinases; D011493:Protein Kinase C