Multiplex Western blots are sensitive and linear, detecting incremental 6 (or 3)% changes in protein levels. Line graphs presenting the relative signal intensity of the indicated protein kinases expressed in 3T3 cells but not in brain (A), plotted against the percentage of 3T3 lysate, or the protein kinases expressed in brain but not in 3T3 cells (B), plotted against the percentage of mouse brain homogenate. Mouse brain homogenate was spiked with incremental 3, 6, 9, or 12%, or 6, 12, 18, or 24% 3T3 cell lysate. The resolved proteins were transferred and probed by multiplex Western blot. The Western blots for CDK1, RSK2, CHK1, CDK4 (A), PKCγ (B) at 6% incremental changes, and p39 (B) at 3% incremental changes, are shown at the bottom of each graph as examples. The regression coefficient (r2) for each protein is indicated.