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Detection of Phosphorylated and Total ERK and p38 MAPK by Bio-Plex Assay, ELISA, and Western Blotting, Rev A

om 12 g/well to 3 g/well (Figure 2A), which suggests that further dilution is necessary to detect a dose response. The Bio-Plex assay showed a distinct dose response (Figure 2B) that correlated well with the western blot (Figure 2C).

Total ERK
Total ERK1/2 was detected by ELISA in HEK 293 lysates with protein concentration ranging from 10 g/well to 0.625 g/well and absorbance ranging from 0.331 to 0.160 (Figure 3A). To explore the Bio-Plex total ERK2 assay sensitivity further, lysates were further diluted to a protein concentration range of 1.250.078 g/well (Figure 3B). In comparison, a faint band was visible on the western blot at a total protein concentration of 0.625 g/lane (Figure 3C).

Both sandwich antibody methods detected total ERK in HeLa lysates with protein concentration ranging from 13 g/well to 0.41 g/well. A flattened lower-end dose response was observed with the ELISA, and an absorbance range of 0.5000.158 showed a limited ability to distinguish between different samples (Figure 3D). The Bio-Plex total ERK2 assay better distinguished signal intensities from the high end to the low end, with MFI from 11,713 to 603 (Figure 3E). The western blot band became faint at 3.25 g/well, indicating the level of sensitivity compared to the Bio-Plex assay (Figure 3F).

Phospho-ERK
Epidermal growth factor (EGF)-treated HEK 293 cell lysates were used for phospho-ERK (Thr202/Tyr204) assay comparison. Lysates were prepared using the Bio-Plex cell lysis kit or by following a protocol provided with the ELISA kit. ELISA showed a limited ability to distinguish the first two samples, with total protein concentrations of 2 g/
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