| Assay Method Information | |
| | pCHK1 Cellular Assay |
| Description: | Inhibitors of ATR kinase are effective at inhibiting the ATR-driven phosphorylation of the downstream target Chk1 kinase at Serine 345, following the addition of 4-nitroquinoline N-oxide, a chemical used to induce DNA damage. Cellular IC50 for the inhibitors of ATR described herein were measured in HT-29 colorectal adenocarcinoma cells. HT-29 cells were routinely maintained in McCoy's 5A media (ATCC Catalog #30-2007) supplemented with 10% fetal bovine serum (Sigma Catalog # F2442) and 1× Penicillin-Streptomycin (Gibco Catalog #15140-122) using a humidified incubator (37° C., 5% CO2, and ambient 02).In preparation for the CHK1 (p-Ser345) ALPHASCREEN SUREFIRE assay, cells were harvested and resuspended in McCoy's 5A media supplemented with 10% fetal bovine serum and 1× Penicillin-Streptomycin. Cells were seeded onto a 384-well black CELLSTAR Tissue Culture Plate (VWR Catalog #89085-314) at a density of 13,000 cells/well in a volume of 40 uL. The microplate was incubated overnight (approximately 20 hours) at 37° C. with 5% CO2 and ambient 02. Stock solutions of the test compounds were prepared in 100% DMSO (Sigma, Catalog # D2650) and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:33 in culture medium, and 10 ul/well were transferred to the tissue culture plate. Following the compound addition the microplate was incubated at 37° C. for 90 minutes. 10 uL of 4-nitroquinoline N-oxide (Sigma Aldrich Catalog # N8141-1G) diluted in media (final concentration 12 uM) were added to the tissue culture plate followed by a 120 minute incubation at 37° C. The cells were then washed with PBS and lysed using 10 uL/well SUREFIRE Kit lysis buffer diluted to 1× in water (PerkinElmer Catalog # TGRCHK1S50K), with mixing on an orbital shaker at 500 rpm for 20 min at RT. Lysates were frozen at −20° C. overnight.4 uL/well of lysate was then transferred from the tissue culture plate to a 384-well, white, low volume, PROXIPLATE (PerkinElmer Catalog #600828). 5 uL/well of the acceptor bead solution, prepared by diluting SUREFIRE Kit activation buffer (PerkinElmer Catalog # TGRCHK1S50K) and ALPHASCREEN Protein A acceptor beads (PerkinElmer Catalog #6760617R) in SUREFIRE Kit reaction buffer (PerkinElmer Catalog # TGRCHK1S50K), were added to the lysates under subdued light and incubated at room temperature for 120 min. 2 uL/well of the donor bead solution, prepared by diluting ALPHASCREEN Streptavidin donor beads (PerkinElmer Catalog #6760617R) in SUREFIRE Kit dilution buffer (PerkinElmer Catalog # TGRCHK1S50K), were added under subdued light and incubated at room temperature for an addition 120 minutes. The pCHK1 ALPHASCREEN signal was measured using an ENVISION plate reader (PerkinElmer). IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |