cAMP Gs dynamic kit
Optimal performance when applied to Gs activation or inhibition
Advanced phospho-ERK (Thr202/Tyr204) cellular kit HTRF®
This HTRF kit is designed for robust quantification of ERK modulation, phosphorylated at Thr202/Tyr204.
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Ease-of-use
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High sensitivity
Overview
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.
Benefits
- VALIDATED ON MULTIPLE CELL TYPES
- SPECIFICITY
- DATA NORMALIZATION
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 proteins phosphorylation state under a no-wash assay format.
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) 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.
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.
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.
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.
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.
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.
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
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).
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.
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.
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 - Notes d'application
Changes in CREB and ERK phosphorylation levels after beta-amyloid treatment - Notes d'application
Investigating kinase activity in a cellular context
HTRF cellular assays - Présentations scientifiaues
In collaboration with the Faculty of health and medical sciences Denmark - Présentations scientifiaues
Open R&D: Sanofi Access Platform
In collaboration with Sanofi - Présentations scientifiaues
In collaboration with the Faculty of health and medical sciences Denmark - Présentations scientifiaues
HTRF: One technology, many uses in a pre-clinical laboratory setting
Benefits and considerations of HTRF - Présentations scientifiaues
HTRF a versatil approach for 7TM drug discovery
In collaboration with MRC - Présentations scientifiaues
Cisbio 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 solutions for NASH - Flyers
HTRF products and supporting scientific content to investifgate GPCRs - Flyers
HTRF solutions for each cell type - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
HTRF assays for Oncology and Inflammation
Signaling in the immune system - Brochures
In collaboration with Molecular Devices - Posters
PI3K/AKT/mTor translational control pathway - Posters
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
Multidimentional GPCR investigations - Posters
TCR signaling investigation with HTRF phospho assays
Study a pathway of interest in PBMC and T cells - Notes d'application
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Notes d'application
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Notes d'application
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Notes d'application
HTRF and Tag-lite technologies in multidimensional cell-based assays for GPCR investigations
4 GPCR challenges, 1 solution - Posters
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Notes techniques
Key success tips to study GPCR signaling
Critical insights into the optimization of ERK phosphorylation assays - Notes techniques
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Notes d'application
Sensitive detection of GPCR-mediated Erk1/2 phosphorylations in various cellular models
4 cellular models: A single GPCR monitoring approach - Notes d'application
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 - Notes techniques
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
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Notes techniques
How to run a cell based phospho HTRF assay
What to expect at the bench - Vidéos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Vidéos
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Présentations scientifiaues
STING HTRF offer to bridge innate and adaptive immunity
cGAS-STING signaling pathway from A to Z - Brochures
All your HTRF assays in one document! - Catalogue
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 - Vidéos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Vidéos
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 - Infographies
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 - Notes d'application
Assays for autoimmunity research
Advance your research on autoimmune diseases - Flyers
Cell-based assays for neurosciences
HTRF solutions - 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. - Notes d'application
a convincing demonstration of the reliability of the AlphaLISA and HTRF KRAS portfolios to evaluate compound in vitro therapeutic profiles in a cellular context - Notes d'application
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques
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 - Notes d'application
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Notes d'application
Podcast: The new era of Neurodegeneration Research
Discover the future of neurodegeneration research - Vidéos
Manual ERK-Advanced Kit / 64aerpeg-64aerpeh
64aerpeg-64aerpeh - Protocoles
Manual ERK-Advanced Kit / 64aerpet
64aerpet - Protocoles
Safety Data Sheet (DEU) ERK-Advanced Kit / 64AERPEG
64AERPEG - Fiches de sécurité
Safety Data Sheet (ELL) ERK-Advanced Kit / 64AERPEG
64AERPEG - Fiches de sécurité
Safety Data Sheet (FRA-FR) ERK-Advanced Kit / 64AERPEG
64AERPEG - Fiches de sécurité
Safety Data Sheet (ITA) ERK-Advanced Kit / 64AERPEG
64AERPEG - Fiches de sécurité
Safety Data Sheet (SPA) ERK-Advanced Kit / 64AERPEG
64AERPEG - Fiches de sécurité
Safety Data Sheet (ENG-GB) ERK-Advanced Kit / 64AERPEG
64AERPEG - Fiches de sécurité
Safety Data Sheet (ENG-US) ERK-Advanced Kit / 64AERPEG
64AERPEG - Fiches de sécurité
Certificate of Analysis ERK-Advanced Kit / 64AERPEH
64AERPEH Lot 18E - Rapports de contrôle qualité
Certificate of Analysis ERK-Advanced Kit / 64AERPEH
64AERPEH Lot 19A - Rapports de contrôle qualité
Safety Data Sheet (DEU) ERK-Advanced Kit / 64AERPEH
64AERPEH - Fiches de sécurité
Safety Data Sheet (ELL) ERK-Advanced Kit / 64AERPEH
64AERPEH - Fiches de sécurité
Safety Data Sheet (FRA-FR) ERK-Advanced Kit / 64AERPEH
64AERPEH - Fiches de sécurité
Safety Data Sheet (ITA) ERK-Advanced Kit / 64AERPEH
64AERPEH - Fiches de sécurité
Safety Data Sheet (SPA) ERK-Advanced Kit / 64AERPEH
64AERPEH - Fiches de sécurité
Safety Data Sheet (ENG-GB) ERK-Advanced Kit / 64AERPEH
64AERPEH - Fiches de sécurité
Safety Data Sheet (ENG-US) ERK-Advanced Kit / 64AERPEH
64AERPEH - Fiches de sécurité
Safety Data Sheet (DEU) ERK-Advanced Kit / 64AERPET
64AERPET - Fiches de sécurité
Safety Data Sheet (ELL) ERK-Advanced Kit / 64AERPET
64AERPET - Fiches de sécurité
Safety Data Sheet (FRA-FR) ERK-Advanced Kit / 64AERPET
64AERPET - Fiches de sécurité
Safety Data Sheet (ITA) ERK-Advanced Kit / 64AERPET
64AERPET - Fiches de sécurité
Safety Data Sheet (SPA) ERK-Advanced Kit / 64AERPET
64AERPET - Fiches de sécurité
Safety Data Sheet (ENG-GB) ERK-Advanced Kit / 64AERPET
64AERPET - Fiches de sécurité
Safety Data Sheet (ENG-US) ERK-Advanced Kit / 64AERPET
64AERPET - Fiches de sécurité
Safety Data Sheet (DEU) ERK-Advanced Kit / 64AERPEY
64AERPEY - Fiches de sécurité
Safety Data Sheet (ELL) ERK-Advanced Kit / 64AERPEY
64AERPEY - Fiches de sécurité
Safety Data Sheet (FRA-FR) ERK-Advanced Kit / 64AERPEY
64AERPEY - Fiches de sécurité
Safety Data Sheet (ITA) ERK-Advanced Kit / 64AERPEY
64AERPEY - Fiches de sécurité
Safety Data Sheet (SPA) ERK-Advanced Kit / 64AERPEY
64AERPEY - Fiches de sécurité
Safety Data Sheet (ENG-GB) ERK-Advanced Kit / 64AERPEY
64AERPEY - Fiches de sécurité
Safety Data Sheet (ENG-US) ERK-Advanced Kit / 64AERPEY
64AERPEY - Fiches de sécurité
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