When intracellular ERK1/2 is activated, phosphorylation occurs at its Thr202 and Tyr204 sites. After cell membrane lysis, endogenous levels can be detected using the Phospho-ERK1/2 (Thr202/Tyr204) Kit. As shown in the figure below, this kit employs a sandwich assay to detect pERK1/2 (Thr202/Tyr204) in samples. The kit contains an antibody labeled with the fluorescent donor Eu, which recognizes p-ERK1/2 (Anti-pERK1/2-Eu), and an antibody labeled with the fluorescent receptor A3, which recognizes ERK1/2 (Anti-ERK1/2-A3). When both antibodies simultaneously bind to pERK1/2 produced after cell lysis, Anti-pERK1/2-Eu and Anti-ERK1/2-A3 come within <10 nm of each other, enabling fluorescence resonance energy transfer (FRET). Excitation of the donor with 320/340 nm light induces emission of 620 nm light, which excites the fluorescent receptor to produce fluorescence at a specific wavelength of 665 nm. The concentration of pERK1/2 (Thr202/Tyr204) in the sample is directly proportional to the FRET signal intensity (the ratio of 665 nm to 620 nm light intensity).

When intracellular ERK1/2 is activated, phosphorylation occurs at its Thr202 and Tyr204 sites. After cell membrane lysis, endogenous levels can be detected using the Phospho-ERK1/2 (Thr202/Tyr204) Kit. As shown in the figure below, this kit employs a sandwich assay to detect pERK1/2 (Thr202/Tyr204) in samples. The kit contains an antibody labeled with the fluorescent donor Eu, which recognizes p-ERK1/2 (Anti-pERK1/2-Eu), and an antibody labeled with the fluorescent receptor A3, which recognizes ERK1/2 (Anti-ERK1/2-A3). When both antibodies simultaneously bind to pERK1/2 produced after cell lysis, Anti-pERK1/2-Eu and Anti-ERK1/2-A3 come within <10 nm of each other, enabling fluorescence resonance energy transfer (FRET). Excitation of the donor with 320/340 nm light induces emission of 620 nm light, which excites the fluorescent receptor to produce fluorescence at a specific wavelength of 665 nm. The concentration of pERK1/2 (Thr202/Tyr204) in the sample is directly proportional to the FRET signal intensity (the ratio of 665 nm to 620 nm light intensity).