The independent channels of the LI-COR MPX (Multiplex) Blotter make it possible to optimize blocking buffer, primary antibody dilution, and secondary antibody dilution on a single Western blot. Western blotting procedures that generate a blot of 7.0 x 8.5 cm are easily adapted to the MPX format. The process fits into any laboratory’s standard Western blot workflow. Both home- made and pre-cast gels can be used to generate blots. Electrophoresis and transfer to nitrocellulose membrane are performed under standard conditions. Clamping the blot into the MPX Blotter creates up to 24 independent channels, allowing different conditions to be tested in each channel. The range of usable channels per sample is relative to comb size. For Western blot optimization, a single-well gel (“prep gel”) is all that is needed. For this application, any detection method can be used, including near-infrared (NIR) fluorescence and chemiluminescence. This document presents general guidelines for use with the Odyssey® family of Infrared Imaging Systems.