Briefly, samples were diluted to the approximately 10μg of total protein (working range for assay was 1–25μg) using provided assay buffer. Additional analyte beads (ERK and β-tublin) were added to the 11 premixed magnetic beads and added to each well in a 96 well plate. Samples and reconstituted control cell lysates (stimulated and unstimulated) were added to the plate and incubated with bead mixture overnight (16–20h) at 2–8°C on a plate shaker (700rpm) protected from light. The plate was washed using a handheld magnet (EMD Millipore Catalog #40–285) to retain the magnetic beads during decanting. Biotinylated detection antibodies were added to the plate and incubated on a shaker at room temperature, protected from light for 1h. Detection antibody was decanted and Streptavidin-Phycoerythrin (SAPE) was added and incubated on a shaker at room temperature, protected from light for 15min. Next, a provided amplification buffer was added and allowed to incubate on a shaker at room temperature, protected from light for 15min. Finally, this solution was decanted, and the beads were suspended in assay buffer before being read using the Luminex® 200
