| Assay Method Information | |
| | Phosphotyrosine (PY) ELISA |
| Description: | Cells used were tumor cell lines naturally expressing high levels of tyrosine kinases. Expression levels at the RNA level were derived from the NCI Developmental Therapeutics Program (NCI-DTP) web site public molecular target information (http://www.dtp.nci.nih.gov/mtargets/mt_index.html). Briefly, cells at 60-75% confluence are placed in serum-free medium for 18 h to reduce the background of phosphorylation. Cells were always >98% viable by Trypan blue exclusion. Cells are then pre-treated for 60 min with various concentrations of test compound, followed by growth factor. The reaction was stopped and cells permeabilized by quickly removing the media from the cells and adding ice-cold Tris-buffered saline (TBS) containing 0.05% triton X-100, protease inhibitor cocktail and tyrosine phosphatase inhibitor cocktail. The TBS solution is then removed and cells fixed to the plate by 30 min at 60 deg C and further incubation in 70% ethanol for an additional 30 min. Cells are further exposed to block (TBS with 1% BSA) for 1 h, washed, and then a horseradish peroxidase (HRP)-conjugated phosphotyrosine antibody added overnight. The antibody was removed; cells are washed again in TBS, exposed to an enhanced luminol ELISA substrate (Pierce Chemical, Rockford, IL, USA) and light emission measured using an UV Products (Upland, CA, USA) BioChemi digital darkroom. Data were graphed as a percent of cells receiving growth factor alone and IC50 values estimated from 2-3 separate experiments (n=8-24) using Prism 5.0. |
| Affinity data for this assay | |
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