PolyGT-Biotin substrate
PolyGT;Biotin is a substrate for tyrosine;specific protein kinases
MAb PT66-Eu cryptate HTRF®
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
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Ease-of-use
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High affinity
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.
Benefits
- NO LIMITATION IN ATP CONCENTRATIONS
- ANY KIND OF SUBSTRATES
- PHOSHO-(TYR) SPECIFIC
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
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
From biochemical to cell based - Brochures
Safety Data Sheet PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Safety Data Sheet (DEU) PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Safety Data Sheet (ELL) PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Safety Data Sheet (FRA-FR) PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Safety Data Sheet (ITA) PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Safety Data Sheet (SPA) PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Safety Data Sheet (ENG-GB) PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Safety Data Sheet (ENG-US) PT66-Eu / 61T66KLA
61T66KLA - Safety Data Sheet
Batch Information PT66-Eu / 20240929
61T66KLA Batch 60B - Batch Information
Batch Information PT66-Eu / 20250323
61T66KLA Batch 61A - Batch Information
Safety Data Sheet PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Safety Data Sheet (DEU) PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Safety Data Sheet (ELL) PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Safety Data Sheet (FRA-FR) PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Safety Data Sheet (ITA) PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Safety Data Sheet (SPA) PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Safety Data Sheet (ENG-GB) PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Safety Data Sheet (ENG-US) PT66-Eu / 61T66KLB
61T66KLB - Safety Data Sheet
Batch Information PT66-Eu / 20230612
61T66KLB Batch 60A - Batch Information
Batch Information PT66-Eu / 20230612
61T66KLB Batch 60B - Batch Information
Batch Information PT66-Eu / 20250323
61T66KLB Batch 61A - Batch Information
Plate Reader Requirement
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