Advanced phospho-ERK (Thr202/Tyr204) cellular kit HTRF®

This HTRF kit is designed for robust quantification of ERK modulation, phosphorylated at Thr202/Tyr204.

See more
  • Ease-of-use Ease-of-use
  • High sensitivity High sensitivity

This HTRF kit is designed for robust quantification of ERK modulation, phosphorylated at Thr202/Tyr204.



The Advanced Phospho-ERK assay kits are designed for the robust and highly sensitive quantification of ERK phosphorylation on Thr202/Tyr204 as an MAPK pathway readout. The simple mix-and-read protocol eliminates all wash steps for faster analysis and high quality output. The Total-ERK1/2 assay is compatible with the buffers from the Phospho or Advanced phospho-ERK kits, so the same lysate can be used for analysis of the total and the phosphorylated protein populations. This kit is optimal for GPCR and RTK targeted screening.



Advanced 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.
Advanced Phospho-ERK (Thr202/Tyr204) assay principle

Advanced Phospho-ERK (Thr202/Tyr204) 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 (Thr202/Tyr204) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Advanced Phospho-ERK (Thr202/Tyr204) 2-plate assay protocol

Advanced Phospho-ERK (Thr202/Tyr204) 1-plate assay protocol

Detection of Phosphorylated ERK (Thr202/Tyr204) 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.
Advanced Phospho-ERK (Thr202/Tyr204) 1-plate assay protocol

Unique sensitivity of Advanced phospho-ERK compared to WB

HEK293 cells were cultured for 48 h. Cells were then stimulated with 50 nM EGF for 5 min. After medium removal and lysis, cell lysates were collected and centrifuged for 10 minutes. Serial dilutions were performed and analyzed side-by-side by Western Blot and by HTRF Advanced phospho-ERK. By using HTRF Advanced phospho-ERK (Thr202/Tyr204) only 300 cells are sufficient for minimal signal detection, while 10,000 cells are needed for a Western Blot signal. The HTRF Advanced phospho-ERK assay is at least 30-fold more sensitive than the Western Blot, and shows optimal correlation.
HTRF Ratio Advanced phospho-ERK kit
HTRF Signal vs WB intensity Advanced Phospho-ERK kit

Basal phospho-ERK dose response for inhibitors without stimulation

Advanced phospho-ERK enables the study of MAPK pathway inhibitor compounds on the basal level of activation, without the need of pathway stimulation. Due to its high cross reactivity, Advanced phospho-ERK is highly compatible with many different model species: human, mouse, rat, dog, monkey, hamster, mink, bovine, pig, D. Melanogaster and zebrafish. All experiments were performed using the two-plate assay protocol for adherent cells. One day after seeding, the cell lines were all treated with their corresponding inhibitor for 30 min at 37°C, 5% CO2.
Dose response of various MAPK pathway inhibitors and their effect on Erk phosphorylation using the Cisbio Avanced Phospho-Erk kit

Phospho-ERK modulation in Diabetes models & patient blood cells

Advanced phospho-ERK assay kit is ideal for monitoring agonist stimulation in pancreatic beta-cell lines or patient PBMCs. The experiments were performed on two pancreatic beta-cell lines: Ins-1E and Min6. The two-plate assay protocol was utilized. Both cell lines were seeded at 70,000 cells/well for 4 days. Cells were washed and incubated with Krebs Ringer Buffer not containing glucose. After 2 hours of starvation, cells were stimulated with glucose. The PBMC's were dispensed at 100,000 cells/well and then stimulated with PMA for 30 min at 37°C, 5% CO2.
Phospho-ERK modulation in Diabetes cell models INS-1 and MIN6

Sensitive phospho-ERK detection in tumor xenografts

BxPC-3 pancreatic tumor xenografts were excised from nude mice, ground and lysed. After centrifugation, the soluble fraction was collected and serial dilutions were performed prior to detection with HTRF Advanced phospho-ERK kit reagents.
Sensitive phospho-ERK detection in BxPC-3 tumor xenografts

Optimized cell density for phospho-ERK detection

Due to the high sensitivity of Advanced phospho-ERK, it is recommended to work with low cell densities of standard cell lines when expecting a high ERK phosphorylation level, to ensure operation in the linear range of the kit and to achieve an optimal S/B. Dose-response experiments were performed on HEK293 cells with the EGFR agonist EGF or the small molecule Raf kinase inhibitor L779-450, using the two-plate assay protocol for adherent cells. Cells were plated at 25,000, 50,000 or 100,000 cells/well for 24h at 37°C, 5% CO2.
Detection of Erk phosphorylation on HEK293 cells stimulated with increasing concentrations of EGF
Dose-response inhibition of Erk phosphorylation in HEK293 cells treated with L779-450

Gaq/i coupled receptor activation

Results obtained on CHO-CCR5 (25 000 cells) activated with Rantes & MIP-Iß for 10', using the two-plate assay protocol of the phospho-ERK cellular assay
CHO-CCR5 activated with Rantes & MIP-Iß and subsequent phsophorylation measured withthe Erk kit

Screening robustness

CHO-M1 (5,000 cells/well - 384-well small volume plate) were stimulated with Carbachol for 10 minutes at RT. Z' values in fifty replicates were calculated between unstimulated cells (basal level) and 2 selected Carbachol concentrations, 1.3 µM and 4 µM, corresponding to the EC80 and EC100 respectively. Assay was performed using the One-plate assay protocol of the phospho-ERK kit on a robotic system from CyBio™ (CyBivario equipped with 384/25 µL pipeting head). Results were read on PHERAStar Plus (BMG Labtech).

Z' screening value obtained using the Cisbio adavanced phospho-erk kit

Total-ERK1/2 assay to control phosphorylation of ERK1/2

A431 cells (100,000 cells/well) were activated with EGF for 10 min, using the two-plate assay protocol of the Phospho-ERK1/2 and Total-ERK1/2 assays. As expected, results obtained show a dose-response increase of ERK1/2 phosphorylation upon EGF stimulation while ERK1/2 expression level remains constant.
dose-response increase of ERK1/2 phosphorylation upon EGF stimulation while ERK1/2 expression level remains constant.

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.

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Analysis of the effect of aggregated beta-Amyloid on cellular signaling pathways critical for memory in Alzheimer's disease

Changes in CREB and ERK phosphorylation levels after beta-amyloid treatment - Application Notes

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Structure, function and pharmacology of G protein-coupled receptors: State-of-the-art and future challenges

In collaboration with the Faculty of health and medical sciences Denmark - Scientific Presentations

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

HTRF assays of IP1, cAMP and pERK pathways employed to study biased signaling of the calcium-sensing receptor

In collaboration with the Faculty of health and medical sciences Denmark - Scientific Presentations

HTRF: One technology, many uses in a pre-clinical laboratory setting

Benefits and considerations of HTRF - Scientific Presentations

HTRF a versatil approach for 7TM drug discovery

In collaboration with MRC - Scientific Presentations

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Biomarker and Cell Signaling Assays for Fibrosis and NASH

HTRF and Alpha solutions for NASH - Flyers

GPCR research from A to Z

HTRF products and supporting scientific content to investifgate GPCRs - Flyers

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the Immune System - Brochures

Deciphering the mechanism of action of drugs targeting EGFR, by Cisbio's advanced cell-based pathway readout assays.

mechanism of action of EGFR inhibitors assessed in the pancreatic BXPC3 cell line - Posters

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

TCR signaling investigation with HTRF phospho assays

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

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

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

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Key success tips to study GPCR signaling

Critical insights into the optimization of ERK phosphorylation assays - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Sensitive detection of GPCR-mediated Erk1/2 phosphorylations in various cellular models

4 cellular models: A single GPCR monitoring approach - Application Notes

Understanding GPCRs is the key to improved DD

In collaboration with GEN - Guides

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

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

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

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

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Molecular basis of neuroinflammation and neurodegeneration diseases

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

Neurodegeneration and its main related diseases

Discover this infographic design on neurodegenerative diseases - Infographics

Neuroinflammation study combining hiPSC-derived astrocytes and HTRF

See published experiments and data demonstrating how immunoassays rise to the challenge of astrocyte studies in neuroinflammation research - Application Notes

Assays for autoimmunity research

Advance your research on autoimmune diseases - Flyers

Assays for neurosciences research

Advance your research on neurodegenerative diseases - Flyers

Inhibition of FGFR Signaling Pathways Studied in Cancer Cell Lines Using HTRF

The knowledge and data necessary to understand the inhibition value of FGFR receptors involved in human cancers. HTRF technology enables the monitoring of the activity of the AZD4547 inhibitor on cell signal transduction in 3 cancer cell lines. - Application Notes

Detection of MAPK activation to evaluate the efficacy and potency of KRAS / SOS1 inhibitors by AlphaLISA and HTRF technologies

a convincing demonstration of the reliability of the AlphaLISA and HTRF KRAS portfolios to evaluate compound in vitro therapeutic profiles in a cellular context - Application Notes

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Podcast: The new era of Neurodegeneration Research

Discover the future of neurodegeneration research - Videos

Manual ERK-Advanced Kit / 64aerpeg-64aerpeh

64aerpeg-64aerpeh - Product Insert

Manual ERK-Advanced Kit / 64aerpet

64aerpet - Product Insert

Certificate of Analysis ERK-Advanced Kit / 64AERPEG

64AERPEG Batch 19C - Quality Control Report

Certificate of Analysis ERK-Advanced Kit / 64AERPEG

64AERPEG Batch 19B - Quality Control Report

Certificate of Analysis ERK-Advanced Kit / 64AERPEG

64AERPEG Batch 19D - Quality Control Report

Certificate of Analysis ERK-Advanced Kit / 64AERPEH

64AERPEH Batch 18E - Quality Control Report

Certificate of Analysis ERK-Advanced Kit / 64AERPEH

64AERPEH Batch 19A - Quality Control Report

Certificate of Analysis ERK-Advanced Kit / 64AERPET

64AERPET Batch 17F - Quality Control Report

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.

Let's find your reader