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

The Anti-phospho threonine-Eu cryptate is a labeled polyclonal antibody. It is highly specific for the presence of phosphothreonine in peptides or proteins and shows no cross-reactivity with the corresponding non-phosphorylated threonine residues. This Anti-Phosphothreonine antibody does not react with phosphotyrosine or phosphoserine. This allows the construction of a highly 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 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 PAb Antiphosphothreonine-Eu cryptate prepared in a buffer containing EDTA to stop the enzymatic reaction.
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