Cells are seeded into a 96-well plate, treated per the experimental protocol, fixed, permeabilized, blocked, and washed.
Primary antibodies from two different host species are added. One is specific for the target protein and one is specific for a normalization protein. Unbound antibody is washed away.
Two species-specific secondary antibodies, labeled with either horseradish peroxidase (HRP) or alkaline phosphatase (AP), are used to detect the specific primary antibody. Unbound secondary antibody is washed away.
Two spectrally distinct fluorogenic substrates for HRP or AP are used to simultaneously detect both target and normalization protein in the same well. Fluorescence is measured with an excitation of 540 nm and emission at 600 nm for the F1 substrate (HRP-specific substrate). A second reading is taken with an excitation of 360 nm and emission at 450 nm for the F2 substrate (AP-specific substrate). Fluorescence of the target protein can be adjusted to the normalization protein to account for well-to-well variations.