title: Immune-complex assays for tyrosine protein kinases.
abstract: Tyrosine protein kinases (TPKs) represent a diverse group of enzymes that contribute to cellular signal transduction. The generally low abundance of TPKs, coupled with their rapid activation and deactivation, usually precludes their purification through conventional biochemical means. Using immune-complex protein kinase assays, the presence or absence of a given TPK can be established and an estimation of its functional state obtained. In the Basic Protocol of this unit, TPKs are immunoprecipitated, allowed to autophosphorylate in the presence of labeled ATP, run out on an SDS-PAGE gel, and detected by autoradiography. Alternate protocols are provided for the assessment of the functional state of TPKs by providing a potential substrate along with the labeled ATP in the reaction mixture. In the first alternate protocol, the exogenous substrate is a protein, permitting simultaneous assessment of autophosphorylation and exogenous substrate phosphorylation. The second alternate protocol utilizes a peptide substrate, resulting in a rapid, high-throughput assay that evaluates only exogenous substrate phosphorylation.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Burkhardt
authors.forename: J B
authors.initials: JB
authors.lastname: Bolen
mesh_terms: D000255:Adenosine Triphosphate / Q000276:immunology; D000818:Animals; D007611:Aprotinin / Q000494:pharmacology; D001345:Autoradiography / Q000379:methods; D002364:Caseins / Q000276:immunology; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D010455:Peptides / Q000276:immunology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D010751:Phosphopyruvate Hydratase / Q000276:immunology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000276:immunology; D011817:Rabbits; D015398:Signal Transduction / Q000276:immunology; D012969:Sodium Fluoride / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
chemical_list: D002364:Caseins; D007976:Leupeptins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D014638:Vanadates; D010664:Phenylmethylsulfonyl Fluoride; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D007611:Aprotinin; D011505:Protein-Tyrosine Kinases; D010751:Phosphopyruvate Hydratase; C032854:leupeptin