| Assay Method Information | |
| | DNA-PK Enzyme-Linked Immunosorbent Assay |
| Description: | On day one, coat 96-well plate (ThermoFisher. Cat #: 442404) with GST-p53 (1-101) peptide (purified by Pharmaron, BCS department) by diluting 3 μg of GST-p53 each well with 0.1 M Na2CO3/NaHCO3, pH 9.6. Incubate the plate overnight at 4° C. The second day, remove coating buffer, wash 2× with PBST (1×PBS containing 0.1% Tween-20). Then add DNA-PK enzyme solution (Invitrogen, #PR9107A; the final DNA-PK concentration is 0.1 μg/mL), series dilution compounds (the final top concentration is 100 nM, 3 fold series dilution, with total 10 doses) and ATP solution (the final ATP concentration is 20 μM) to the 96-well plate. Incubate the plate at 25° C. for 1 hour. Then wash 3× with PBST (1×PBS containing 0.1% Tween-20). Block the plate with PBST+ 1% BSA at 4° C. overnight. The third day, wash 4× with PBST (1×PBS containing 0.1% Tween-20). Then add Phospho-p53 primary antibody (cell signaling Technology, #9286, Phospho-p53 (Ser15) (16G8) Mouse mAb) (1/1000) to each well. Seal with plate and incubate the plate for 1 h at 37° C. Wash 4× with PBST (1×PBS containing 0.1% Tween-20), add 100 μL of HRP-linked secondary antibody (Cell signaling Technology, #7076, Anti-mouse IgG, HRP-linked Antibody) (1/1000) to each well. Seal with tape and incubate the plate for 30 min at 37° C. Wash 4× with PBST (1×PBS containing 0.1% Tween-20), add 100 μL of TMB (Cell signaling Technology, #7004) substrate to each well. Seal with tape and incubate the plate for 10 min at 37° C. Then add 100 μL of Stop solution (Cell signaling Technology, #7002) to each well. Read the plate at 450 nm to detect absorption. |
| Affinity data for this assay | |
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