Methods for Measuring Real Time Kinase Activity
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example 1
Quantification of p38 Kinase Activity
[0240] A mouse monoclonal antibody specific for p38 kinase was attached to the wells of a 96-well plate. The concentrations of p38 antibody in carbonate buffer were 3 μg / ml, 6 μg / ml and 12 μg / ml and 100 μl of each concentration was used per well.
[0241] Murine macrophage cells (RAW264.7) were exposed to anisomycine and incubated for about 2 hours in culture. Control cells were untreated. After incubation, cell culture was removed from the cells and the cells were washed. Cells were then lysed using commercially available cell lysis reagents, and the cell lysate was added to the wells of the coated plate.
[0242] After the binding agent was allowed to capture the specific kinase of interest (p38), the kinase activity sensor was added to the wells (50 ng) along with 1 mM ATP. In this instance, the kinase recognition motif of the activity sensor comprised the amino acid sequence: AHLQRLSI(dP) (SEQ ID NO. 134), where the serine was the phosphorylatio...
example 2
Quantification of Erk1 / 2 Kinase Activity
[0243] A rabbit monoclonal antibody specific for Erk1 / 2 kinase was attached to the wells of a 96-well plate. The concentrations of Erk1 / 2 antibody in carbonate buffer were 3 μg / ml, 6 μg / ml and 12 μg / ml and 100 μl of each concentration was used per well.
[0244] Murine embryonic fibroblast cells (3T3) were exposed to platelet derived growth factor (PDGF) and incubated for about 2 hours in culture. Control cells were untreated. After incubation, cell culture was removed from the cells and the cells were washed. Cells were then lysed using commercially available cell lysis reagents, and the cell lysate was added to the wells of the coated plate.
[0245] After the binding agent was allowed to capture the specific kinase of interest (Erk1 / 2), the kinase activity sensor was added to the wells (50 ng) along with 1 mM ATP. In this instance, the kinase recognition motif of the activity sensor comprised the amino acid sequence: AHLQRLSI(dP) (SEQ ID NO. 1...
example 3
Measurement of Akt1 Activity in Crude Lysates from PDGF-Treated or Control NIH3T3 Cells
[0246] NIH3T3 cells were seeded in 100 mm dishes and grown in DMEM plus 10% fetal bovine serum until 90% confluent. The cells were then incubated overnight in serum-free medium to induce quiescence, followed by treatment with PDGF A / B (50 ng / mL, 10 min) or control media. Cell lysates were prepared and Akt1 activity was assayed as described below in the assay procedure.
[0247] The PDGF-treated sample showed a reaction rate of 1.13 RFU / sec, whereas the control treated sample had a markedly reduced rate of 0.12 RFU / sec, resulting in a dramatic signal-to-noise ratio of 9. Other assay control groups (bead only group or bead and antibody only group) also had reaction rates less than 0.12 RFU / sec. Activity was assayed as described in the Assay Procedure section below using SEQ ID NO. 25. Results are shown in FIG. 3.
Assay Procedure:
Reagents:
[0248] Wash Buffer (prepare 1× stock): Dilute an appropriat...
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