Phospho-ERK (Thr202/Tyr204), Advanced phospho-ERK (Thr202/Tyr204), and Total ERK1/2 Cellular Assay Kits

The trusted homogeneous cell-based assays for GPCR and RTK targeted screening as MAPK pathway readout

Fast track your research and speed up your time to consistent results - from basic research to high throughput drug screening. Cisbio’s homogeneous HTRF® phospho-ERK assay kits are designed for the cell-based quantitative detection of ERK modulation, phosphorylated on Thr202/Tyr204. The simple mix-and-read protocol eliminates all wash steps for a faster analysis and easy miniaturization while maintaining a high quality and specific output.

Ideal for ERK normalization – the total-ERK1/2 assay is compatible with the buffers from the Phospho-ERK or Advanced phospho-ERK kits, so the same lysate can be used for analysis of the total and the phosphorylated protein populations.

   Check our lysis buffer compatibility      Check our species compatibility

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Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total ERK1/2assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-ERK1/2 antibodies, one labeled with Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with ERK1/2, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-protein can be quantitatively detected using the HTRF phospho- and total cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well small volume assay plate where the HTRF reagents are added. This also enables monitoring the cells' viability and confluence in an appropriate cell culture plate.

One-plate protocol

Detection of phosphorylated ERK with HTRF® reagents is performed in a single plate used for plating, stimulation, lysis and detection. No washing steps are required. This protocol, designed for HTS, enables miniaturization while maintaining a high quality output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Guidelines and tips to get the best out of your assays

Guidelines for cell culture and Lysis to prior detection, Download tehcnical note

Download our cell-based phospho-protein data normalization application note

Simplified Pathway

MAPK/ERK cell signaling

The MAPK/ERK signaling cascade is activated by a wide variety of receptors involved in growth and differentiation including growth factor receptors, like receptor tyrosine kinases RTKs, integrins, cytokine receptors, GPCRs  and ion channels. The core cascade Ras-Raf-MEK-ERK signal transduction leads to the regulation of a great variety of cellular processes including adhesion, cycle progression, migration, apoptosis/survival, differentiation, metabolism, proliferation, etc. ERK is a Mitogen-Activated Protein kinase that belongs to the family of serine/threonine kinases. MEK1/2 catalyze the phosphorylation of ERK1/2 at Tyr204 and then Thr202. Both phosphorylations are required for ERK1/2 activation. In response, ERK phosphorylates hundreds of cytoplasmic and nuclear substrates, including regulatory molecules and transcription factors. The wide complexity and diversity of MAPK signaling makes ERK a key regulator and major signaling node in biology. Signal transduction via MAPK/ERK is a highly conserved pathway, with extensive cross talk between other pathways and a wide variety of signal and stimuli responsiveness known today. More than 80 pathways in which ERK is involved are currently described (KEGG database). The activity of the Ras-Raf-MEK-ERK core cascade is increased in about one-third of all human cancers. Thus, inhibition of targets in the MAPK/ERK pathway represents an important anti-tumor strategy, whereas the assessment of ERK activation itself serves as a valuable ubiquitous pathway readout. Phospho-ERK is therefore an important tool for monitoring upstream dose-dependent modulation of endogenous or overexpressed targets.

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 cyclic AMP, diacylglycerol, inositol 1,4,5 trisphosphate and Ca2+, but also mediate. ERK1/2 phosphorylation. Activation of Gai/o, Gas, Gaq/11 or Ga12/13 modulates ERK1/2 activation via numerous mechanisms. In addition, both alpha and beta subunits of G proteins can stimulate ERK1/2 phosphorylation through transactivation of receptor tyrosine kinases. GPCRs have also been shown to mediate ERK1/2 activation in a G protein-independent but beta-arrestin dependent manner.

Product Performances

1. Unique sensitivity of Advanced phospho-ERK and optimal correlation to Western Blot

HEK293 cells were grown in a T175 flask at 37°C, 5% CO2 for 48 h. Cells were then stimulated with 50 nM EGF for 5 min. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer #1 for 30 min at room temperature. Soluble fractions were then collected after 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer #1 and 16 µL of each dilution were dispensed 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

2. Basal phospho-ERK dose response readouts for inhibitors without prior cell stimulation

Advanced phospho-ERK enables the study of MAPK pathway inhibitor compounds on the basal level of activation, without the need of pathway stimulation. The illustration shows dose response evaluations of various MAPK pathway inhibitors on several unstimulated model cell lines from different species.

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.

3. Phospho-ERK modulation in Diabetes models & patient blood cells

Advanced phospho-ERK is the ideal assay to monitor agonist stimulation in pancreatic beta-cell lines or patient PBMCs. The experiments were performed on two pancreatic beta-cell lines, Ins-1E and Min6, applying the two-plate assay protocol for adherent cells. Both cell lines were seeded at 70,000 cells/well for 4 days. Cells were washed and incubated with Krebs Ringer Buffer without glucose. After 2 h starvation, the cells were stimulated with glucose: 5 min on Min6 or 10 min on Ins-1E at 37°C, 5% CO2.

For the peripheral blood mononuclear cells (PBMC) the two-plate assay protocol for suspension cells was applied. The PBMCs were dispensed at 100,000 cells/well and then stimulated with PMA for 30 min at 37°C, 5% CO2.

4. Sensitive phospho-ERK detection in tumor xenografts

BxPC-3 pancreatic tumor xenografts were excised from nude mice, ground and lysed. After centrifugation of the lysate, the soluble fraction was collected and serial dilutions were performed before dispensing 16 µL of each dilution into the detection plate and adding the HTRF Advanced phospho-ERK detection reagents.

5. Optimized cell density for phospho-ERK detection in standard cell lines with high ERK phosphorylation

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.

Cells were stimulated with increasing concentrations of EGF for 5 min at 37°C, 5% CO2.

Cells were firstly treated with a dose-response of L779-450 for 30 min, and then stimulated with 2 nM EGF for 5 min 

6. Gαq/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

7. 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™ (CyBi®vario equipped with 384/25 µL pipeting head). Results were read on PHERAStar Plus (BMG Labtech).

8. Validation of the total-ERK assay on various species of cell lines: human, mouse and hamster

Human (Hela, A431, HEK293, Jurkat), murine (NIH 3T3) and Hamster (CHO-K1) cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging. ERK1/2 was detected using 16µL of cell lysate. Cell lines from other species have not been tested, hence they must be evaluated case by case.

9. Total-ERK1/2 assay used to control the phosphorylation status 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Advanced ERK phospho-T202 /Y204 kit - 500 tests64AERPEG
Advanced ERK phospho-T202 /Y204 kit - 10,000 tests64AERPEH
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Advanced ERK phospho-T202 /Y204 kit - 1 x 96 tests64AERPET
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Advanced ERK phospho-T202 /Y204 kit - 50,000 tests64AERPEY
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ERK phospho-T202/Y204 kit - 500 tests64ERKPEG
ERK phospho-T202/Y204 kit - 10,000 tests64ERKPEH
ERK phospho-T202/Y204 kit - 50,000 tests64ERKPEY
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ERK total kit - 500 tests64NRKPEG
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ERK total kit - 10,000 tests64NRKPEH
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ERK total kit - 1 x 96 tests64NRKPET
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ERK total kit - 50,000 tests64NRKPEY
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Companion products

DescriptionCat. noProduct insertMSDS
ERK phospho-T202/Y204 kit control lysate62ERKTDA
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Advanced ERK phospho-T202 /Y204 kit control lysate64AERTDA
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ERK total kit control lysate64NRKTDA
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