| Assay Method Information | |
| | DNA-PK Enzyme-Linked Immunosorbent Assay |
| Description: | On day one, a 96-well plate (ThermoFisher, Cat #: 442404) was coated with GST-p53 (1-101) peptide (purified by Pharmaron, BCS department) by diluting 3 μg of GST-p53 in each well with 0.1 M Na2CO3/NaHCO3 (pH 9.6). The plate was incubated overnight at 4° C. On the second day, the coating buffer was removed, and the plate was washed twice with PBST (1×PBS containing 0.1% Tween-20). The DNA-PK enzyme solution (Invitrogen, #PR9107A; the final DNA-PK concentration: 0.1 μg/mL) was then added. The compounds were serially diluted to the final maximal concentration of 100 nM (3 fold series dilution, a total of 10 doses), and an ATP solution (the final ATP concentration: 20 μM) was added to the plate. Incubate the plate at 25° C. for 1 hour. The plate was washed three times with PBST (1×PBS containing 0.1% Tween-20) and blocked with a solution of PBST and 1% BSA at 4° C. overnight. The third day, the plate was washed four times with PBST (1×PBS containing 0.1% Tween-20). Anti-phospho-p53 primary antibody (cell signaling Technology, #9286, Phospho-p53 (Ser15) (16G8) Mouse mAb) (1/1000) was added to each well. The plate was sealed, incubated 1 h at 37° C., and washed four times with PBST (1×PBS containing 0.1% Tween-20). An HRP-linked secondary antibody (Cell signaling Technology, #7076, Anti-mouse IgG, HRP-linked Antibody) (1/1000) (100 μL) was added to each well. The plate was sealed with tape, incubated 30 min at 37° C., and washed four times with PBST (1×PBS containing 0.1% Tween-20). At this time, 100 μL of TMB (Cell signaling Technology, #7004) substrate were added to each well. The plate was sealed with tape and incubated the plate 10 min at 37° C. Stop solution (Cell signaling Technology, #7002) (100 μL) was added to each well, and the plate was subjected to the absorption detection at 450 nm. |
| Affinity data for this assay | |
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