反应详情

Bead-Based Kinase Assays. SwellGel Discs (Pierce) were suspended in cold 50 mM Tris, pH 7.5 so that 1 μl of bead suspension bound 1 μg of GST fusion protein. One nmol of GST-Crkl substrate was incubated with the glutathione bead suspension for 1 h at 4° C. with constant rotation. The substrate-bound beads were washed twice with ice-cold 50 mM Tris-HCl, pH 7.5 containing 10 mM MgCl2. Substrate-bound beads were then incubated with either recombinant v-Abl or K562 cell lysate. The v-Abl reaction mixtures contained: 20 μl 4×buffer (200 mM Tris-HCl, 40 mM MgCl2, 4 mM DTT, pH 7.5); 20 μl 40 μM ATP; 0.5 μl v-Abl (EMD Biosciences, Inc., San Diego, Calif.); 0 or 20 μl 400 μM imatinib; and water to a total volume of 80 μl. The K562 cell lysate reaction mixtures contained: 20 μl 4×buffer; 20 μl 40 μM ATP; 50 μg K562 cell lysate; 0 or 20 μl 400 μM imatinib; and water to a total volume of 80 μl. The reactions allowed to proceed for 1 h at 30° C. Following the reaction, the beads were washed twice with ice-cold 50 mM Tris, pH7.5. GST-Crkl substrates were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. Kinase assay samples were loaded in a 12% SDS-PAGE gel and transferred to nitrocellulose membranes according to standard procedures. Consistent sample loading was verified using the Memcode Reversible Protein Stain Kit (Pierce). Membranes were probed with anti-phosphotyrosine antibodies.

1. customfor 1 h
2. customat 4° C.
3. washThe substrate-bound beads were washed twice with ice-cold 50 mM Tris-HCl, pH 7.5
4. additioncontaining 10 mM MgCl2
5. customThe v-Abl reaction
6. customThe K562 cell lysate reaction
7. customthe reaction
8. washthe beads were washed twice with ice-cold 50 mM Tris, pH7.5
9. washGST-Crkl substrates were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0