The mitogen-activated protein kinase (MAPK/ERK) pathway plays a key role in cell proliferation, differentiation and migration. Stimulation by mitogens eventually leads to phosphorylation of ERK1 (T202/Y204) and ERK2 (T185/Y187). The MAPK/ERK cascade presents many interesting drug targets for the development of cancer therapies.Cell-based ELISA measures dually phosphorylated ERK1/2 in whole cells. This simple and efficient assay eliminates the need for cell lysate preparation and can be used to study kinase signaling and the effects of kinase inhibitors on cells. In this assay, cells are grown in 96- well plates and treated with ligands or drugs. Cells are then fixed and permeabilized in the wells. ERK1/2 phosphorylation (pERK) is measured using a double immunoenzymatic labeling procedure.Key Features:Safe. Non-radioactive assay.Simple and convenient. Total and pERK can be measured in the same sample.Applications:Determination of ERK phosphorylation status in whole cells.Evalutation of effects of ligands or drugs on ERK phosphorylation.Species tested: human, mouse, rat.Kit Contents:10X Wash Buffer: 25ml Blocking Buffer: 25mlALP Substrate: 6ml HRP Substrate: 6mlERK-Ab1: 10ul pERK-Ab1 10ulHRP-Ab2: 10ul ALP-Ab2 10ulStorage Conditions:Store all reagents at -20°C. Shelf life of 6 months after receipt.Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.Materials Required But Not Provided:37% formaldehyde; 3% H2O2; black cell culture 96-well plate; plate sealers; deionized or distilled water; pipetting devices; cell culture incubators; centrifuge tubes; fluorescence plate reader capable of reading at lex/em=535/590nm and at lex/em =360/450nm.
