| Assay Method Information | |
| | In Vitro Screen for ALK5 Kinase Inhibition |
| Description: | In a 96 well filter-bottom plate (Millipore, #MSDV N6B 50), 58 μL Assay Buffer is added to reach well. Add 10 μL of Cold ATP mix in Assay Buffer, then 10 μL of a 1:10 dilution of α-Casein stock. Then add 2 μL of compound being tested (DMSO) at a 50× final concentration. Hot ATP mix (10 μL) is added, and the reaction is started with the addition of 10 μL of a 1:350 dilution of the ALK5 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 μL 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 μL ice-cold 10% TCA. The plate bottom is blotted before and after removing plastic sub-base, and dried overnight at room temperature. Add 30 μL 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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