b'Overview of detection methodsWith a variety of detection techniques to choose covering chemiluminescence, fluorescence, or chromogenic detection, you can select a technology that matches your experimental requirements and equipment available (Table 16). Table 16. Comparison of western blot detection methods.Chemiluminescence Fluorescence ChromogenicMajor advantage Highest sensitivity Detect multiple targets at once Easily visualize blots without equipmentTechnology overview Enhanced chemiluminescentSimultaneous detection of multipleDirect visualization of your target (ECL) HRP and AP substratesproteins on the same blot using aprotein using color detection reagentsproviding pictogram- tocombination of primary antibodies and femtogram-level detection associated fluorophore-conjugated secondary antibodiesSignal source Indirect signal from enzymatic reaction Direct signal from fluorophore Indirect signal from enzymatic reactionSignal duration Limited (hours) Extended (weeks to months) Extended (weeks to months)SensitivityExcellent Good Limited, best for high-abundance proteinsConsistency Possible variation between blots,High reproducibility between blots Possible variation between blotswhich can be mitigated by using high-duration substratesDetection X-ray film and imaging instruments Imaging instruments capable ofVisualno instrumentation requiredexciting fluorophores and detecting the subsequent light emissionOther considerations Stripping and reprobing ofCare is needed to avoidStripping and reprobing of blot blot possible fluorescence contamination not possibleLong exposure times possible, asAntibody selection becomesBest method for high-abundance no excitation light source required tomore challenging as the degreeproteins and when imaging or capture signal of multiplexing increases (to avoidfilm processing instrumentation is antibody cross-reactivity) not available42'