Phospho-4EBP1 (T37/T46) + Total 4EBP1 TR-FRET Cellular Assay Kit

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Product Name Phospho-4EBP1 (T37/T46) + Total 4EBP1 TR-FRET Cellular Assay Kit
Species Human
Description The Phospho-4EBP1 (T37/T46) & Total 4EBP1 assay kit is a homogeneous time resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay (Figure 1). The THUNDERTM 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-4EBP1 (T37/T46) & Total 4EBP1 in the cell lysates are detected in separate wells with two FR-Ab2 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-4EBP1 (T37/T46) & Total 4EBP1 in the cell lysate. Data can be expressed as either the signal at 665 nm or the 665 nm/615 nm ratio.

Sandwich TR-FRET immunoassay for semi-quantitative detection of Total AKT pan

This assay kit measures intracellular levels of Phospho-4EBP1 (T37/T46) & Total 4EBP1 protein in cell lysates using a simple, rapid and sensitive immunoassay based on the homogeneous (no-wash) THUNDERTM TR-FRET technology. The kit is compatible with both adherent and suspension cells.

Background The Phospho-4EBP1 (T37/T46) & Total 4EBP1 assay kit is a homogeneous time resolved Förster resonance energy transfer (TR-FRET) sandwich immunoassay (Figure 1). The THUNDERTM 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-4EBP1 (T37/T46) & Total 4EBP1 in the cell lysates are detected in separate wells with two FR-Ab2 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-4EBP1 (T37/T46) & Total 4EBP1 in the cell lysate. Data can be expressed as either the signal at 665 nm or the 665 nm/615 nm ratio.
Method TR-FRET
Sample Types Cell lysates (adherent or suspension cells)
Gene ID 1978
SwissProt Q13541
Storage at -80°C
Intended Use For research use only
Configuration Components (500 test):
Eu-labeled Phospho-4EBP1 (T37/T46) & Total 4EBP1 antibody (Eu-Ab1) (20 µL)
Acceptor-labeled Phospho-4EBP1 (T37/T46) & Total 4EBP1 antibody (FR-Ab2) (80 µL)

Eu-labeledtotal-AKTpanantibody61(5Enum-Ab3) (5 µL)

Acceptor-labeled total AKT pan antibody (FR-Ab4) (20 µL)
5X Lysis Buffer 2 (5 mL)
10X TR-FRET Detection buffer (250µL)
Postive control lysate (500 µL)

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