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Total MEK1 cellular kit HTRF®

The total MEK1 kit quantifies cellular endogenous MEK1 when studying the MAPK/ERK pathway, and can be used as a normalization assay for the phospho-MEK1 kit.
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  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • Ready-to-use Ready-to-use
The total MEK1 kit quantifies cellular endogenous MEK1 when studying the MAPK/ERK pathway, and can be used as a normalization assay for the phospho-MEK1 kit.
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Overview

The HTRF total MEK1 assay kit is designed for the quantitative detection of total MEK1, phosphorylated and unphosphorylated, to normalize the phosphorylation status of MEK1. It is the ideal assay for studying the RAS-RAF-MEK cascade in the MAPK/ERK pathway. Phosphorylated by RAF, this dual specificity mitogen-activated protein kinase is responsible for the phosphorylation of ERK, one of the most important signaling nodes, and protein kinase in cell proliferation, survival, and differentiation pathways.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-MEK1 assay principle

The Total-MEK1 assay quantifies the expression level of MEK1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-MEK1 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of MEK1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total-MEK1 assay principle

Total-MEK1 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 Total MEK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total MEK1 2-plate assay protocol

Total-MEK-1-plate assay protocol

Detection of total MEK 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.
Total-MEK 1-plate assay protocol

Validation of the total-MEK1 assay on various cell lines

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, 3 mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging, total MEK1 was detected using 16 µL of cell lysate. Cell lines from other species have not been tested, hence they must be evaluated case by case.
Validation of the total-MEK1 assay on various cell lines

Total-MEK1 assay used to control the phosphorylation status of MEK1

Hela cells (100,000 cells/well) were activated with EGF for 5 min, using the two-plate assay protocol of the Phospho-MEK1 (Ser218/222) and Total-MEK1 assays. Results obtained show a dose-response increase of MEK1 phosphorylation upon EGF stimulation while MEK1 expression level remains constant.
Total-MEK1 assay used to control the phosphorylation status of MEK1

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

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

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - 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

GPCR research from A to Z

HTRF products and supporting scientific content to investifgate GPCRs - Flyers

Species compatibility

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

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

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

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

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - 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

Product Insert MEK1 total Kit / 64NE1PEG-64NE1PEH

64NE1PEG-64NE1PEH - Product Insert

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

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

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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