Revvity, 61GT0BLA, HTRF Anti-PolyGT Biotin Antibody, 5,000 Assay Points

Poly-(Glu,Tyr) (4:1) is a random copolymer which has been shown to be a substrate for tyrosine-specific protein kinases. Poly-(GT)-biotin may be used with Streptavidin-XL665 and Anti-phosphotyrosine-cryptate for easily optimized HTRF® tyrosine kinase assays. HTRF enables to perform assay at physiological ATP concentrations. HTRF assays offer many advantages over other technologies: Homogeneous add-and-read format No wash steps Low background Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates Stable signal, providing flexibility in time of readout or size of assays
Assay principles HTRF Tyrosine kinase assay use typically a combination of a Europium-cryptate labeled anti-phosphoresidue antibody, SA-XL665 and PolyGT-Biotin as substrate. The signal is proportional to the concentration of phospho-tyrosine. Assay protocol HTRF tyrosine kinase assay have two basic phases: 1. Enzymatic step: biotinylated PolyGT 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 PolyGT is then detected by the addition of Streptavidin-XL665 and a Europium-cryptate labeled anti-phosphotyrosine antibody (MAb PY20-Eu cryptate or MAb PT66-Eu cryptate) prepared in a buffer containing EDTA to stop the enzymatic reaction.