In-cell ELISAs for measuring relative total MAPK3/1 and phospho-MAPK3/1 levels.

The Thermo Scientific Pierce ERK1/2 In-Cell ELISA Kits enable the relative quantitation of total and phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) in whole cells using a 96- or 384-well microplate format. The 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.

Highlights of the In-Cell ELISA Kits:

• Two reliable detection formats available: (Learn more)
  • 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

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.

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.

Related Products and Links:
ELISA reagent reservoirs and sealing tape
Browse all In-Cell ELISA Kits for specific targets
Basic In-Cell ELISA Detection Kits – basic kit to be used with your primary antibodies

Product #	Description	Pkg. Size
62206	ERK1/2 Colorimetric In-Cell ELISA Kit Sufficient materials for 1 x 96 wells Includes: Blocking Buffer, 50 ml 20X Tris Buffered Saline, 50 ml Surfact-Amps 20 (10% Tween-20 Detergent), 10 ml Surfact-Amps X-100 (10% Triton X-100 Detergent), 10 ml HRP Conjugate, 0.14 ml TMB Substrate, 58 ml TMB Stop Solution, 55 ml Janus Green Whole-Cell Stain, 50 ml Elution Buffer, 2 x 30 ml Thin Plate Seal Assembly, 8 each Anti-Phospho ERK 1 & 2 (Thr 202/Tyr 204) Antibody Anti-ERK1/2 Antibody	kit
62211	ERK1/2 Near Infrared In-Cell ELISA Kit Sufficient materials for 1 x 96 wells Includes: Blocking Buffer, 100 ml 20X Tris Buffered Saline, 50 ml Surfact-Amps 20 (10% Tween-20 Detergent), 10 ml Surfact-Amps X-100 (10% Triton X-100 Detergent), 10 ml DyLight 680 Goat Anti-Mouse IgG (H+L), 55 µl DyLight 800 Goat Anti-Rabbit IgG (H+L), 55 µl Thin Plate Seal Assembly, 8 each Anti-Phospho ERK 1 & 2 (Thr 202/Tyr 204) Antibody Anti-ERK1/2 Antibody	kit