Microplate assays to measure total and phospho-ERK1/2 in whole cells.
Thermo Scientific Pierce ERK1/2 In-Cell ELISA Kits are colorimetric and fluorescent microplate assays optimized to measure total and phosphorylated extracellular signal-regulated kinase 1 and 2 (phospho-ERK1/2) in whole cells.
These ELISA kits for whole cells complete kits include a mouse monoclonal anti-ERK1/2 antibody, rabbit monoclonal anti-phospho-(Thr202/Tyr204) ERK1/2 antibody, detection reagents and all buffers required for either colorimetric or near infrared detection. The ERK1/2 In-Cell ELISA Kits enable simultaneous detection of both total and phospho-(Thr202/Tyr204) ERK1/2.
Highlights:
Measures total and phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in whole cells using a 96- or 384-well microplates
colorimetric detection kit for use with standard microplate readers
near infrared fluorescent detection kit for use with IR imaging system
Whole-cell detection requires no cell lysis, protein extraction or special sample preparation
Provides better data and higher throughput than quantitative Western blotting
Less costly (one well vs. one lane) than Western blotting
Product Details:
Stimulation with EGF causes a 6-fold increase in the level of MAPK3/1 phosphorylation in HeLa cells. Relative total and phospho-MAPK3/1 levels were measured in HeLa cells using the ERK1/2 Colorimetric In-Cell ELISA Kit. Cells were serum-starved overnight and stimulated with 100ng/ml EFG for five minutes immediately prior to fixation.The level of MAPK3/1 phosphorylation increased approximately 6-fold in response to EGF stimulation where as total MAPK3/1 were not significantly affected. Note: Absolute absorbance values cannot be directly compared between phospho and total MAPK3/1 experiments because they use antibodies with different sensitivities.
Treatment with U0126 inhibits EGF-induced phosphorylation of MAPK3/1 in HeLa cells. Relative total and phospho-MAPK3/1 levels were measured in HeLa cells using the ERK1/2 Colorimetric In-Cell ELISA Kit. Cells were serum-starved overnight, treated with U0126 for two hours and then stimulated with 100ng/ml EFG for five minutes immediately prior to fixation. At 10µM U0126, the phosphorylation of MAPK3/1 was inhibited to levels similar to that observed in HeLa cells that were not stimulated with EGF (data not shown). Note: Absolute absorbance values cannot be directly compared between phospho and total MAPK3/1 experiments because they use antibodies with different sensitivities.
Overview of ERK1/2 and phosphorylation of ERK1/2:
ERK1 and ERK2 are also referred to as mitogen-activated protein kinase 3 and 1, respectively (MAPK3, MAPK1) and regulate a broad range of cellular processes including gene transcription, cytoskeletal organization, metabolic homeostasis, cell growth, and apoptosis. Within the MAPK/ERK signal transduction pathway, extracellular stimuli may be transmitted into the cell through a receptor tyrosine kinase (RTK) and relayed through MAPKK kinase phosphorylation of MAPK kinase which, in turn, phosphorylates MAPK family members. Dual phosphorylation of ERK1/2 on thr 202 and tyr 204 causes translocation into the cell nucleus and phosphorylation of transcriptional regulators. Disregulation of ERK1/2 is implicated in cancer phenotypes. The ERK1/2 In-Cell ELISA Kit enables simultaneous detection of both total and phospho-(Thr202/Tyr204) ERK1/2.
