THUNDER™ Phospho-AKT pan (T308) TR-FRET Cell Signaling Assay Kit | KIT-AKTT308P

This assay kit measures intracellular levels of phospho-AKT pan (T308) protein in cell lysates using a simple, rapid and sensitive immunoassay based on the homogeneous (no-wash) THUNDER™ TR-FRET technology. The kit is compatible with both adherent and suspension cells.

SPECIFICITY
This assay kit contains two specific and selective antibodies, one that recognizes AKT pan phosphorylated at Thr308 and another that recognizes an invariant epitope of AKT pan.

SPECIES REACTIVITY
Human; Mouse (Swiss-Prot Acc. P31749, P31751, Q9Y243; Entrez Gene Id 207, 208 and 10000). Other species should be tested on a case-by-case basis.

TR-FRET ASSAY PRINCIPLE
The Phospho-AKT pan (T308) assay kit is a homogeneous timeresolved Förster resonance energy transfer (TR-FRET) sandwich
immunoassay (Figure 1). The THUNDER™ Cell Signaling assay workflow consists of 3 steps (Figure 2). Following cell treatment, cells are first lysed with the specific Lysis Buffer provided in the kit.
Then Phospho-AKT pan (T308) in the cell lysates is detected with a pair of fluorophore-labeled antibodies in a simple "add incubatemeasure" 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 (FRAb2). 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 Phospho-AKT pan (T308) in the cell lysate. Data can be expressed as either the signal at 665 nm or the 665 nm/615 nm ratio.