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 IRDye® 800CW or IRDye® 680LT are used to detect the specific primary antibody. Unbound secondary antibody is washed away.
The plate is imaged by scanning simultaneously at 700 nm and 800 nm with an infrared imaging system, such as LI-COR® Odyssey® Infrared Imaging System. Fluorescent signals derived from the target protein can be normalized to those derived from the second protein to correct for well-to-well variations.
LI-COR, Odyssey, and IRDye are registered trademarks of LI-COR, Inc.
*This product is covered by US 6,995,274; US 7,504,089; foreign equivalents and patents pending.