| Assay Method Information | |
| | Kinase Assay |
| Description: | In vitro kinase assays were carried out by using recombinant murine c-abl containing SH3, SH2 and kinase domains (residues 46-531) and full length immunoprecipitated Bcr-abl. Recombinant abl was expressed in Sf9 insect cells and purified as described above. Bcr-abl immune complexes were obtained with Ab-3 anti-abl monoclonal antibody (Oncogene Science) from Ba/F3.p210 lysates as previously described. 1 μg of recombinant abl or immunoprecipitated Bcr-abl (from 3×106 cells) were incubated with various concentrations of test compound (0.1, 1 and 10 M) in kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM EDTA, 1 mM DTT, 0.015% Brij 35), 100 μM ATP and 1 μCi [γ-32P]-ATP for 30 min at 30° C. (Calbiochem buffer and protocol). The reaction was stopped by addition of Laemmli buffer and the proteins were resolved by SDS-PAGE in a 4-20% gel. The phosphoproteins were visualized by autoradiography and the autophosphorylation quantitated using a phosphoimager (STORM, Molecular Devices). |
| Affinity data for this assay | |
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