ABSTRACT
With recent advances in techniques for detecting chemiluminescent substrates, hybridization with nonisotopic rather than radiolabeled probes is becoming more common. In the Basic Protocol, nylon membranes carrying transferred nucleic acids are prepared for hybridization with biotinylated probes by UV cross-linking. This is a critical step in the procedure and the Support Protocol provides a detailed description of light-source calibration. After hybridization, the target nucleic acid is detected through a series of steps that lead to an enzyme-catalyzed light reaction. The Alternate Protocol describes chemiluminescent detection based upon antibody recognition of digoxigenin-labeled probes. For both biotinylated and digoxigenin-labeled probes, chemiluminescent detection is more sensitive than colorimetric detection and has the added advantage that the membrane can be used for multiple film exposures, then stripped and redetected with different probes.
