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Assay Method Information |
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| | ALK-5 Kinase Assay |
| Description: | In a 96 well filter-bottom plate (Millipore, #MSDV N6B 50), 58 ul Assay Buffer (50 mM HEPES, pH 7.6, with 10 mM NaCl, 10 mM MgCl2, and 1 mM DTT ) is added to reach well. Add 10 ul of Cold ATP mix in Assay Buffer, then 10 ul of a 1:10 dilution of alpha-Casein stock. Then add 2 ul of compound being tested (DMSO) at a 50x final concentration. Hot ATP mix (10 ul) is added, and the reaction is started with the addition of 10 ul of a 1:350 dilution of the ALK-5 protein (2 nM final) in Assay Buffer with 0.05% BSA (Bovine Serum Albumin). The reaction is mixed for 5 minutes at room temperature, and then continued for 145 minutes at room temperature. The reaction is then stopped with the addition of 100 ul of ice-cold 20% TCA (trichloroacetic acid). The assay is then incubated for at least 1 hour at 4° C., and then the contents of each well are filtered by suction through the filter. The wells are washed three times with 200 ul ice-cold 10% TCA. The plate bottom is blotted before and after removing plastic sub-base, and dried overnight at room temperature. Add 30 ul of scintillation fluid, and count 1 minute per well on a Wallac Tri-Lux scintillation counter. |
| Affinity data for this assay | |
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