PAb Anti-phospho PKA substrate-Eu cryptate enables the detection of phosphorylation of peptides or proteins on Ser/Thr in a biochemical kinase assay format.
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  • No-wash
  • Ease-of-use
  • High affinity
PAb Anti-phospho PKA substrate-Eu cryptate enables the detection of phosphorylation of peptides or proteins on Ser/Thr in a biochemical kinase assay format.
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Overview

The Anti-phospho PKA-Eu cryptate is a labeled polyclonal antibody enabling the detection of peptides and proteins containing a phosphorylated Ser/Thr residue with Arginine in the –3 position. It does not detect the non-phosphorylated PKA. This allows the construction of a highly specific biochemical kinase assay. Any kind of substrates can be used, from small synthetic peptides, to natural substrates for a high assay detection versatility. As HTRF tolerates extremely high ATP concentrations, an assay can be performed 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 assay 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 PKA substrate-Eu cryptate , prepared in a buffer containing EDTA to stop the enzymatic reaction.
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