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Feasibility of Multiplexing Bio-Plex Total Target and Phosphoprotein Assays

lexed with phosphoprotein assays?
Can a total target assay detect differences in protein concentration among samples?


Methods
We designed the study using HEK 293 cells, which respond to UV treatment by increasing the level of phosphorylation of several proteins. All proteins selected for the phosphoprotein assays in this study are activated by this treatment. The multiplex run (6-plex) included the following targets:

Total ERK2
Phospho-p53 (Ser15)
Phospho-p70 S6 kinase (Thr421/Ser424)
Phospho-ATF-2 (Thr71)
Phospho-JNK (Thr183/Tyr185)
Phospho-c-Jun (Ser63)

Untreated and UV-treated HEK 293 cell lysates were prepared according to methods detailed in the Bio-Plex phosphoprotein assay instruction manual. Both untreated HEK 293 and UVtreated HEK 293 cell lysates were tested at three different protein concentrations to test dose-response characteristics of the assays. Blank wells were included in the run, with cell lysis/assay buffer used to replace the cell lysate. Duplicates were run for each sample.


Results and Discussion
Total Target Assays Can Be Multiplexed With Phosphoprotein Assays for Distinct Targets
Data from a multiplex run of five phosphoprotein assays with a total ERK2 assay are presented in Figure 1. To show the results clearly, each phosphoprotein signal was plotted together with the total ERK2 assay signal. Although the phosphorylation levels found between UV-treated and untreated cell lysates varied, the total ERK2 signal level at the same protein concentration was very consistent bet
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