| Assay Method Information | |
| | Biochemical Phospho c-Kit Inhibition Assay |
| Description: | For the PathScan phospho c-kit (Tyr719) sandwich ELISA: M-07e cells were resuspended at 2×106 cells/mL in phenol red free, serum-free, GM-CSF-free Iscove's Modified Dulbecco's Medium (IMDM) media with 1% Penicillin-Streptomycin. The cells were then dispensed into the wells of a U-bottom, 96-well plate at 50 uL per well using a multichannel pipet. The plate was allowed to incubate at 37° C. in a humidified tissue culture incubator for 4 hours.After 4 hours of incubation, each well was dosed with 6.25 uL of test compound at a final DMSO concentration of 0.25% for 60 min at 37° C. to generate an 8-point dose concentration series of test compounds in duplicate. Cells were then incubated for 1 hour at 37° C. in a humidified tissue culture incubator. Next, human SCF at 500 ng/mL was added to the appropriate wells at 6.25 uL/well and the plate was shaken at 450 rpm at room temperature for 10 minutes. AlphaLISA 5× Lysis Buffer supplemented with 1× protease and phosphatase inhibitor was then added at 16 uL/well. The plate was sealed with an adherent cover and shaken at 4° C. at 600 rpm for 30 minutes. After this period of lysis, the plate was stored at −80° C. When the 96-well plate containing M07e lysates was ready to be processed, the plate was brought to room temperature and 40 μl from each well was transferred to the wells of a PathScan Phospho-c-Kit (Tyr719) Sandwich ELISA plate (CST, Catalog #7298). The ELISA plate was sealed with an adherent cover and incubated for 2 hours at 37° C. Next, the wells were washed 4 times with the provided 1× Wash Buffer, and 100 uL/well of reconstituted Detection Antibody was added to each well, the plate sealed with an adherent cover, and incubated at 37° C. for 1 hour. The wash procedure was repeated and 100 uL/well of reconstituted HRP-Linked secondary antibody was added. The plate was sealed with an adherent cover and incubated at 37° C. for 1 hour. The wash procedure was repeated and 100 uL/well of TMB Substrate was added. The plate was incubated for 10 minutes at room temperature or until the positive reaction elicited a blue color in the appropriate wells. 100 uL/well of STOP solution was added and shaken gently for a few seconds a microplate reader or conventional spectrophotometer e.g., a Perkin Elmer Envision multimode plate reader, part #2105-0010. |
| Affinity data for this assay | |
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