Revvity, 61P02KAE, HTRF PAb Anti-phospho AKT substrate-Eu cryptate

Overview
The Anti-phospho AKT substrate-Eu cryptate is a labeled polyclonal antibody enabling the detection of Akt when phosphorylated at Ser or Thr preceded by arginine or lysine in -5 and -3 positions. It does not cross-react with phosphorylated Ser or Thr in different configurations (slight cross reactions with -2 and +2 positions). It does not detect the non-phosphorylated Akt. 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.
How it works
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 Anti-phospho AKT substrate-Eu cryptate , prepared in a buffer containing EDTA to stop the enzymatic reaction.