MAb PT66-Eu cryptate

MAb PT66-Eu cryptate enables the rapid detection of phosphorylation of peptides or proteins on Tyrosine in a biochemical kinase assay format.
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  • No-wash
  • Ease-of-use
  • High affinity
MAb PT66-Eu cryptate enables the rapid detection of phosphorylation of peptides or proteins on Tyrosine in a biochemical kinase assay format.
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Overview

PT66-Eu cryptate is a labeled mouse monoclonal IgG1. It shows to react specifically with phosphorylated tyrosine, both as free amino acid or when conjugated to carriers. No cross-reactivity has been observed with non-phosphorylated tyrosine, phosphothreonine, phosphoserine, AMP or ATP. This allows the construction of a highly specific biochemical Tyrosine 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 assays typically use 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 MAb PT66-Eu cryptate prepared in a buffer containing EDTA to stop the enzymatic reaction.

Detection at Physiological ATP Concentration

ATP concentration is an important factor for in vitro kinase assay design, as the sensitivity of ATP competitive inhibitors varies with ATP concentration. As shown in Figure 5, HTRF tolerates the extremely high ATP concentrations (up to mM range) needed for mechanistic studies, e.g. the confirmation of ATP competitive inhibitors. The shift observed for increasing ATP concentrations in the graph confirms the ATP-competitive effect of the compound tested. Assay was performed on a tyrosine kinase revealed by PT66.

Membrane friendly

In addition to fresh and frozen cells, the HTRF Kinase toolbox can be used with membrane preparations. The graph depicts an assay scheme for measuring autophosphorylation of a receptor tyrosine kinase (RTK), using membrane preparations as a source of active kinase.Autophosphorylation of an RTK expressed as a tagged fusion protein is detected using a Eu3+ labeled anti-phospho tyrosine antibody and XL665 labeled anti-tag antibody.
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 PT66-Eu / 61T66KLA-61T66KLB

61T66KLA-61T66KLB - 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

Safety Data Sheet PT66-Eu / 61T66KLA

61T66KLA - Safety Data Sheet

Safety Data Sheet PT66-Eu / 61T66KLB

61T66KLB - Safety Data Sheet

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