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Phospho-ERK (Thr202/Tyr204) cellular kit HTRF®

The phospho-ERK (Thr202/Tyr204) kit measures ERK protein phophorylated at Thr202/Tyr204 as a readout of MAPK pathway activation.
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  • Highly specific Highly specific
  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity
The phospho-ERK (Thr202/Tyr204) kit measures ERK protein phophorylated at Thr202/Tyr204 as a readout of MAPK pathway activation.
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Overview

Phospho-ERK assay kits are designed for the robust quantification of ERK modulation, phosphorylated on Thr202/Tyr204, for GPCR and RTK targeted screening as a MAPK Pathway readout. The simple mix-and-read protocol eliminates all wash steps for faster analysis and high quality output.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • SPECIFICITY

Phospho-ERK (Thr202/Tyr204) assay principle

The Phospho-ERK (Thr202/Tyr204) assay measures ERK when phosphorylated at Thr202/Tyr204. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-ERK (Thr202/Tyr204) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-ERK (Thr202/Tyr204) assay principle

Phospho-ERK 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-ERK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-ERK 2-plate assay protocol

Phospho-ERK 1-plate assay protocol

Detection of Phosphorylated ERK with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-ERK 1-plate assay protocol

MAPK/ERK cell signaling

The MAPK/ERK signaling cascade is activated by a wide variety of receptors involved in growth and differentiation. The core signal transduction cascade elicits regulation of numerous cellular processes including adhesion, migration, apoptosis, differentiation and metabolism. MEK1/2 catalyze the phosphorylation of ERK1/2 at Tyr204 and Thr202. In response, ERK phosphorylates hundreds of cytoplasmic and nuclear substrates. The wide complexity and diversity of MAPK signaling makes ERK a key regulator and major signaling node in biology.
MAPK/ERK cell signaling pathway

ERK in GPCR signaling

ERK modulation plays a major role in GPCR signaling and is therefore an important measurable GPCR readout. GPCRs act via G proteins to regulate a wide range of cellular functions. Upon stimulation, these receptors activate effectors like adenylate cyclase and phospholipase C, which influence not only intracellular concentrations of second messengers like cAMP and Ca2+, but also mediate ERK1/2 phosphorylation. GPCRs have also been shown to mediate ERK1/2 activation in a G protein-independent but beta-arrestin dependent manner.

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Cisbio lysis buffer compatibility

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HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

GPCR research from A to Z

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Species compatibility

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HTRF assays for Oncology and Inflammation

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Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

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Study a pathway of interest in PBMC and T cells - Application Notes

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HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

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Insider Tips for successful sample treatment - Technical Notes

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Understanding GPCRs is the key to improved DD

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HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

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HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

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HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

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Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

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3' video to set up your Phospho assay - Videos

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The essential guide for extending your knowledge on the molecular mechanisms of neurodegenerative diseases - Guides

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Discover this infographic design on neurodegenerative diseases - Infographics

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New insight into neuroinflammation research - Videos

Assays for autoimmunity research

Advance your research on autoimmune diseases - Flyers

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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