Automated nonisotopic assay for protein-tyrosine kinase and protein-tyrosine phosphatase activities.
A sensitive, automated, and nonisotopic assay for protein-tyrosine kinases and phosphatases has been developed. The assay uses commercially available antiphosphotyrosine monoclonal antibodies and the recently developed particle concentration immunofluorescence immunoassay technology. The assay is specific for phosphotyrosine residues, can be performed faster, and is at least 100-fold more sensitive than the current standard filter type radioassay. Myelin basic protein and a synthetic peptide corresponding to the autophosphorylation site of p56lck performed equally well in the detection of p56lck kinase activity. Myelin basic protein phosphorylated on tyrosine residues by p56lck was successfully used as substrate in the detection of phosphatase activity and vanadate or molybdate were shown to inhibit the phosphatase activity. The assay is particularly useful for the rapid detection of enzyme activities in column fractions from biochemical procedures steps and also for screening of large numbers of potential inhibitors or activators of protein-tyrosine kinases and phosphatases.
Babcook
J
J
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
Watts
J
J
Aebersold
R
R
Ziltener
H J
HJ
D000911:Antibodies, Monoclonal; D002852:Chromatography, Ion Exchange; D016231:Fluorescence Polarization Immunoassay; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D004676:Myelin Basic Protein / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D004676:Myelin Basic Protein; D010455:Peptides; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017027:Protein Tyrosine Phosphatases