THUNDER™ Phospho-AKT pan (S473) + Total AKT pan TR-FRET Cell Signaling Assay Kit

The Phospho-AKT pan (S473) + Total AKT pan 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 Phospho-AKT pan (S473) and Total AKT pan in the cell lysates are detected in separate wells with two pairs of fluorophore-labeled antibodies in a simple "add-incubate-measure" format (single-step reagent addition; no wash steps). For detection of the phosphorylated protein, one antibody is labeled with a donor fluorophore (Europium chelate; Eu-Ab1) and the second with a far-red acceptor fluorophore (FR-Ab2). The same approach is used for the second antibody pair detecting the total protein (Eu-Ab3 and FR-Ab4). The binding of the two matched labeled antibodies to distinct epitopes on the target protein (either phospho-AKT pan or total AKT pan) 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 Phospho-AKT pan (S473) and Total AKT pan in the cell lysate. Data can be expressed as either the signal at 665 nm or the 665 nm/615 nm ratio.

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