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Feasibility of Multiplexing Bio-Plex Total Target and Phosphoprotein Assays

ween treatments, indicating that none of the phosphoprotein assays interfered with the total target assay in this multiplex run.

For all tested phosphoprotein targets, the level of detected phosphorylation increased in HEK 293 cells in response to UV treatment, with the extent of increase depending on the phosphoprotein, as expected. The most dramatic signal increase was detected for phospho-c-Jun (Ser63) (Figure 1E).

At 0.6 g/ml protein, there was more than a 40-fold increase in phospho-c-Jun signal detected in UV-treated HEK 293 compared to untreated cell lysate. Notably higher levels of phosphorylation were also detected for phosphorylated p70 S6 kinase and phospho-ATF-2 assays in UV-treated cell lysates. The signals for untreated cell lysates were consistently low across all diluted samples. The low signals, around 60 70 MFI, in untreated cell lysates made the ratio of UV-treated to untreated as high as 76 at 12 g of total protein concentration. Lower phosphorylation levels were detected for phospho-p53 (Ser15) and phospho-JNK (Thr183/Tyr185), but the signal differences between untreated and UV-treated cell lysates were still significant at all tested protein concentrations.

Data from this 6-plex run (one total target assay multiplexed with five phosphoprotein assays) suggest that it is practical to mutiplex Bio-Plex total target assays with Bio-Plex phosphoprotein assays, provided that the targets are not the same.

Total ERK2 Assay Results Correlated With Protein Concentration
The total ERK2 assay showed consistent results in both untreated and UV-treated HEK 293 cell lysates. The signal for total ERK2, though not linear, showed a
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