wash-out experiment
The binding kinetics of each compound to its target is definitive for its biological activity. In a wash-out experiment, the cells are treated with the compound for different durations and then are washed out by PBS. Then the cells are further cultured for different duration in fresh culture media to recover. The readout can be the proliferation rate or the change in the signaling pathway.
Recovery of EGFR autophosphorylation after treatment with GW572016, ZD-1839, and OSI-774. Logarithmically growing HN5 cells were treated with 1 μmol/L inhibitor in culture media for 4 hours. The media was removed, cells were washed twice, and fresh compound-free media was added. The cells were lysed at the indicated time after inhibitor washout, and EGFR was isolated by immunoprecipitation. A, tyrosine phosphorylated EGFR (pEGFR) was determined by Western blot using antiphosphotyrosine antibody. Lysates used for immunoprecipitation were prepared from cells treated with the following agent: Lane 1, DMSO control; Lane 2, GW572016; Lane 3, ZD-1839; and Lane 4, OSI-774. Total EGFR was measured from the same immunoprecipitate samples by Western blot using anti-EGFR antibody. B, The level of Tyrosine-phosphorylated EGFR was quantified for each condition and expressed as the percentage of vehicle-treated sample. The results represent the mean value of three independent experiments; bars, ±SD.
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