PAb Anti-phospho MEK1/2-Eu cryptate

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
  • Ease-of-use
  • 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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Overview

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.

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.

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

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
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