( sites of phosphorylation ide...)Application of the Bio-Plex Cell Lysis Kit in Western Blot Analysis of Phosphoproteins, Rev A

Quantitation of Phosphoproteins
The western blot signals for each phosphoprotein were quantitated (AfCS protocol PP00000007) and normalized to the signal for G protein b subunit (conventional antibody, not phosphospecific) to account for lane-to-lane variability. Fold change in phosphoprotein signal was calculated relative to the average of triplicate zero time points (fold change defined as 1).

Results and Discussion
The protein concentration was 40% greater for WEHI-231 cell extracts prepared using the AfCS protocol than those prepared with the Bio-Plex cell lysis kit (1.4 0.1 vs. 1.0 0.1 mg/ml, respectively, n = 15). Protein from AfCS protocol extracts (12 g) or Bio-Plex cell lysis kit extracts (6 g) was analyzed by multiplex western blotting to quantitate fold changes in protein phosphorylation. Figure 1 shows examples of multiplex western blots with protein migrations identified. The signals for selected proteins were quantitated as fold changes in ligand-stimulated phosphorylation (Figure 2). It is satisfying to find that the magnitude of fold changes in phosphorylation quantitated for the Bio-Plex cell lysis kit extracts was greater than or equal to that calculated for our routine AfCS protocol extracts. We conclude that the Bio-Plex cell lysis method is adaptable to western blotting procedures for phosphoprote
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