PAb Anti-phospho MEK1/2-Eu cryptate HTRF®

PAb Anti-phospho MEK1/2-Eu cryptate enables the rapid detection of MEK1/2 phosphorylation on Ser 217/221 in a biochemical kinase assay format.
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  • No-wash No-wash
  • Ease-of-use Ease-of-use
  • High affinity High affinity
PAb Anti-phospho MEK1/2-Eu cryptate enables the rapid detection of MEK1/2 phosphorylation on Ser 217/221 in a biochemical kinase assay format.
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The Anti-phospho MEK1/2 substrate-Eu cryptate is a labeled polyclonal antibody enabling the detection of MEK1/2 when phosphorylated at Ser217/221. It recognizes singly phosphorylated MEK1/2 at Ser 217 but not MEK1/2 singly phosphorylated at Ser 221. It does not detect the non-phosphorylated MEK1/2. This allows the construction of a specific biochemical kinase assay. Any kind of substrate can be used, from small synthetic peptides to natural substrates, meaning high assay detection versatility. HTRF allows to perform assay at physiological ATP concentrations.



Assay principle

HTRF kinase assay use typically a combination of a Europium-cryptate labeled anti-phosphoresidue antibody, SA-XL665 and a biotinylated* substrate. The signal is proportional to the concentration of phospho-residues. *Alternatively, other tags such as 6HIS, GST, c-myc, DNP, or FLAG may be used to label the kinase substrate.
HTRF kinase assay principle

Assay protocol

HTRF kinase assays have two basic phases: 1. Enzymatic step: biotinylated substrate is incubated with the kinase of interest and with the compounds in presence of co-factors. The reaction starts with the addition of ATP. 2. Detection step: The phosphorylated biotinylated substrate is then detected by the addition of Streptavidin-XL665 and PAb Anti-phospho MEK1/2-Eu cryptate , prepared in a buffer containing EDTA to stop the enzymatic reaction.
HTRF kinase assay protocol

Working with natural ligand, development of COT assay

As shown here, the HTRF Kinase toolbox reagents can be used with any tagged peptide or protein substrates. The performances of each format were compared to P33 filter-binding assay in the study published by J. Jia (Anal. Biochem. 2006:305:268-276 ). In format 1, anti-GST-XL665 and PAb Anti-phospho MEK1/2-Eu cryptate were used to detect the whole GST-tagged protein, while in format 2, a small biotinylated MEK1 peptide was detected using Streptavidin-XL665 and the same phospho specific labeled antibody. Results are published in the table below
HTRF COT assay using 2 different direct formats

Apparent ATP Km values determined by different COT assay formats

Assay Format Apparent ATP Km(µM)
COT/MBP protein direct33P 384 ± 55
COT/MEK protein direct HTRF 279 ± 40
COT/MEK peptide direct HTRF 268 ± 21

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Experience the freedom of building your own assay

HTRF kinase activity toolbox reagents - Flyers

Product Insert A-P MEK1/2-Eu / 61P17KAE-61P17KAZ-61P17KAY

61P17KAE-61P17KAZ-61P17KAY - 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

The essential white paper to expand your knowledge on kinases

Learn about cutting edge kinase research in this free white paper - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Revolutionize your kinases investigations with HTRF

Watch this video to learn more about kinases investigations - Videos

Easy method for kinetic binding determination

How to revolutionize your kinetic binding demonstration with HTRF kinase binding platform assays - Application Notes

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