ECF™ Western is a highly sensitive system for chemifluorescent detection of Western blots analyzed by Image Master™ VDS-CL that allows quantification in even very low target applications. ECF Western blotting permits immunodetection of proteins after separation by SDS-PAGE and following electroblotting onto a PVDF membrane. The system can have standard or enhanced formats. The standard system uses a primary antibody directed towards the target protein, followed by incubation with an anti-species alkaline phosphatase-linked antibody. The enhanced system uses a primary antibody against the target protein, followed by incubation with a second anti-species flourescein-linked antibody. The signal is enhanced by the use of a third antibody with an anti-fluorescent alkaline phosphatase conjugate. The alkaline phosphatase catalyses the conversion of an ECF substrate to a highly fluorescent product that fluoresces strongly at 540–560 nm when the blots are illuminated with UV light (maximum excitation at 430 nm). Analysis and quantification of Western blots is carried out using ImageMaster VDS-CL and ImageMaster 1D Elite software. The use of this non-radioactive method means safe handling and reliable, highly sensitive detection of target proteins.