Enzymes such as HRP or AP convert several substrates to a colored precipitate. As the precipitate accumulates on the blot, a colored signal develops. The reaction can be monitored and stopped when the desired signal over background is observed. Colorimetric detection is easier to perform than film-based chemiluminescence detection; however, the method's single endpoint result does not allow the multiple exposures of chemiluminescence methods. Colorimetric detection is typically considered a medium-sensitivity method compared to radioactive or chemiluminescence detection.
Bio-Rad's AP-based colorimetric kits utilize 5-bromo-4-chloro-3-indolyl phosphate/Nitroblue Tetrazolium (BCIP/NBT), which reacts with the alkaline phosphatase to form a color precipitate for detection. The Immun-Blot AP assay kit includes all necessary reagents for colorimetric detection and can detect up to 100 pg of protein.
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