The Total STAT5 assay kit is a homogeneous time-resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay. The THUNDER™ Cell Signaling assay workflow consists of 3 steps. Following cell treatment, cells are first lysed with the specific Lysis Buffer provided in the kit. Then Total STAT5 in the cell lysates is detected with a pair of fluorophore-labeled antibodies in a simple "add-incubate-measure" format (single-step reagent addition; no wash steps). One antibody is labeled with a donor fluorophore (Europium chelate; Eu-Ab1) and the second with a far-red acceptor fluorophore (FR-Ab2). The binding of the two labeled antibodies to distinct epitopes on the target protein takes place in solution and brings the two dyes into close proximity. Excitation of the donor Europium chelate molecules with a flash lamp (320 or 340 nm) or a laser (337 nm) triggers a FRET from the donor to the acceptor molecules, which in turn emit a TR-FRET signal at 665 nm. Residual energy from the Eu chelate generates light at 615 nm. The signal at 665 nm is proportional to the concentration of Total STAT5 in the cell lysate. Data can be expressed as either the signal at 665 nm or the 665 nm/615 nm ratio.WARNING This product is not for human or veterinary use.
