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

arget assays (summarized in the flowchart opposite). The procedure provided in the ELISA kit vendors manual was followed for the phospho- and total protein ELISAs. The same antibodies specific to each target were used in both western blot and Bio-Plex phosphoprotein and total target assays. The antibodies used in the ELISAs were those provided in the vendor kit.


Results and Discussion
Total p38 MAPK
Total p38 MAPK was detected by ELISA in HEK 293 lysates with protein concentration ranging from 20 g/well to 0.625 g/well and absorbance ranging from 2.623 to 0.317 (Figure 1A). The limit of detection appears to be around 0.625 g/well. A similar dose response was observed with the Bio-Plex assay; however, an MFI value of 897 at the lowest data point (0.625 g/well) suggests the potential to detect further-diluted samples (Figure 1B). The data also suggest high-end saturation over 10 g/well. With the western blot, the target was detected down to a protein concentration of 1.25 g/lane (Figure 1C).

Total p38 MAPK was not detected, nor was a dose response observed with ELISA of HeLa lysates, even at the highest protein concentration tested (Figure 1D). This target was, however, detected with the Bio-Plex assay in each sample, with the MFI ranging from 4,950 to 321 (Figure 1E), which correlated well with the western blot (Figure 1F).

Phospho-p38 MAPK
Phospho-p38 MAPK (Thr180/Tyr182) was detected by ELISA in UV-treated HEK 293 lysate with protein concentration ranging from 12 g/well to 0.75 g/well (Figure 2A). The ELISA displayed a high-end saturation plateau in samples with a protein concentration fr
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