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Research Area
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Description
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Malignant melanoma,Colorectal cancer |
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Protocol
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Kinase Assay
[1]
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| RAF kinase activity measurements |
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 Assay
[2]
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| Cell Lines |
MALME-3M, Colo829, Colo38, A375, SK-MEL28, and A2058 cells |
| Concentrations |
0–10 μM , dissolved in DMSO |
| Incubation Time |
5 days |
| Methods |
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 Study
[2]
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| Animal Models |
Mice (athymic nude) xenograft models of LOX, Colo829, and A375 cells |
| Formulation |
Formulated as microprecipitated bulk powder (MBP), suspended at the desired concentration in an aqueous vehicle containing 2% Klucel LF, and adjusted to pH 4 with dilute HCl |
| Doses |
12.5 mg/kg–100 mg/kg |
| Administration |
Oral gavage twice daily |
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References |
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[1] Bollag G, et al. Nature, 2010, 467(7315), 596-599.
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[2] Yang H, et al. Cancer Res, 2010, 70(13), 5518-5527.
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[3] Prahallad A, et al. Nature, 2012, 483(7387), 100-103.
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[4] Kumar A, et al. J Mol Biol, 2005, 348(1), 183-193.
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