Practice question

Bioluminescent tagging employs luciferase enzymes that generate photons through substrate oxidation without requiring external illumination, thereby avoiding autofluorescence and photobleaching. Firefly luciferase uses D-luciferin, ATP, magnesium, and molecular oxygen to produce light near 560 nm. In blotting context, target protein fused to luciferase or detected by luciferase-conjugated secondary antibody emits light captured by sensitive detectors. Alkaline phosphatase with BCIP/NBT and horseradish peroxidase with TMB produce colorimetric precipitates, while green fluorescent protein requires excitation by blue light to fluoresce. Luciferase therefore provides true bioluminescent detection with high sensitivity.