Kinase Assay: |
Nature,2010 Sep 30;467(7315):596-9. | Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma.[Pubmed: 20823850] | The kinase activities of wild-type RAF and mutants are determined by measuring phosphorylation of biotinylated-BAD protein. For each enzyme (0.01 ng), 20 μL reactions are carried out in 20 mM Hepes (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (v/v) Tween-20, 50 nM biotin-BAD protein, and 1 mM ATP at room temperature. Reactions are stopped at 5 min with 5 μL of a solution containing 20 mM Hepes (pH 7.0), 200 mM NaCl, 80 mM EDTA, 0.3% (w/v) bovine serum albumin (BSA). The stop solution also includes phospho-BAD (Ser112) antibody, streptavidin-coated donor beads, and protein A acceptor beads. The antibody and beads are pre-incubated in stop solution in the dark at room temperature for 30 min. The final dilution of antibody is 1/2000 and the final concentration of each bead is 10 μg/mL. The assay plates are incubated at room temperature for one hour and then are read on a PerkinElmer AlphaQuest reader. Mutant activities are the average of two different batches of purified protein assayed in duplicate in three different experiments. |
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Cell Research: |
Cancer Res,2010 Jul 1;70(13):5518-27. | RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models.[Pubmed: 20551065] | Cell lines:MALME-3M, Colo829, Colo38, A375, SK-MEL28, and A2058 cells
Concentrations: 0–10 μM , dissolved in DMSO
Incubation Time: 5 days
Method:
Cellular proliferation is evaluated by MTT assay. Briefly, cells are plated in 96-well microtiter plates at a density of 1000 to 5000 cells per well in a volume of 180 μL. PLX4032 is prepared at 10 times the final assay concentration in media containing 1% DMSO. Twenty-four hours after cell plating, 20 μL of the appropriate dilution of PLX4032 are added to plates in duplicate. The plates are assayed for proliferation 6 days after the cells are plated. Percent inhibition is calculated and the IC50 is determined from the regression of a plot of the logarithm of the concentration versus percent inhibition.
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Animal Research: |
Cancer Res, 2010 Jul 1;70(13):5518-27. | RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models.[Pubmed: 20551065] | Animal Models: Mice (athymic nude) xenograft models of LOX, Colo829, and A375 cells
Dosages:12.5 mg/kg–100 mg/kg
Administration: Oral gavage twice daily
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