LY2857785 Inhibitor

Solid. Powder

98.00%

Store at -20 degree C for 3 years powder
-80 degree C for 2 years in solvent

448.6

DMSO: 10mg/mL (22.29mM) Need ultrasonic
(<1mg/ml refers to the product slightly soluble or insoluble)

C26H36N6O

CC(C)c1c(cc(cc2)-c3ccnc(N[C@H](CC4)CC[C@@H]4NC4CCOCC4)n3)c2nn1C

1619903-54-6

Solid tumor cells are plated in poly-D-lysine coated and hematologic cell lines are seeded in noncoated 96-well plates overnight before being treated with LY2857785. Solid tumor cells are fixed with Prefer for 20 minutes at room temperature and permeated with 0.1% Triton X-100 in PBS for 15 minutes. Caspase-3 expression is measured by immunofluorescence with anti-activated caspase-3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) activity is measured with In Situ Cell Death Detection Kit. Both assays are analyzed on Acumen Explorer laser-scanning fluorescence microplate cytometer. Hematologic tumor cells are assayed for cell viability with CellTiter-Glo Luminescent Cell Viability Assay.

CDK7 and CDK9 reaction mixtures contain 10mM Tris-HCl (pH 7.4), 10mM HEPES, 5mM DTT, 10uM ATP, 0.5uCi 33p-ATP, 10mM MnCl2, 150mM NaCl, 0.01% Triton X-100, 2% DMSO, 0.05mM CDK7/9ptide, and 2nM CDK7/Mat1/cyclin H, or 2nM CDK9/cyclin T1, respectively. CDK8/cyclin C reaction is performed in HEPES 30mM, DTT 2mM, MgCl2 5mM, 0.015% Triton X-100, 5uM ATP, and 400nM of RBER-CHKStide containing 20nM of enzyme. LY2857785 in DMSO is diluted serially 1:3 for dose-response. Reactions are carried out in 96-well polystyrene plates. The reactions are incubated at room temperature for 60 minutes and followed by termination with 10% H3PO4 or 10% trichloroacetic acid (TCA). For the filter binding assay, reactions are transferred to 96-well filter plates and measured by Microbeta scintillation counter. For ADP Transcreener Fluorescent Polarization Assays, reactions are quenched with ADP detection mix, incubated 2 hours at room temperature and then FP is measured at lambdaex=610nM, lambdaem=670nM on a Tecan Ultra 384 plate reader. The concentration of ADP product is calculated from millipolarization (uP) using a prepared ADP/ATP dilution series as a standard curve. Kinase profiling are carried out in 96-well polystyrene plates. Briefly, in a final volume of 25uL the enzyme is incubated with the appropriate buffer, peptide substrate, and the diluted LY2857785. Reactions are initiated by the addition of ATP/[33P] and the ATP mix is incubated at room temperature for 40 minutes. Reactions are quenched with the addition of 5uL of 3% phosphoric acid, 10uL of the reaction are spotted onto a filter mat, washed 3 times for 5 minutes in 75mM phosphoric acid and once in methanol. Once the filters are dry, they are submitted to scintillation counting.

Cell Cycle/Checkpoint; CDK