washed and exposed to film for hours or days. Radioactive blots can more quickly be detected using storage phosphor plates instead of film; the plates are read on a specialized scanning instru- ment. detailed discussions about the features and benefits of detection by film and scanners are included in Chapter 14, Nucleic Acid Hybridization to indicate the presence of bound antibody The simplest st Enzymatic reactions are used in a number of different systems enzymatic detection is chromogenic. Here the secondary reagent is conjugated to an enzyme, either horseradish peroxidase (HRP) or alkaline phosphatase(AP). After incubation with the sec- ondary reagent and washing, the blot is incubated with a substrate. The enzyme catalyzes a reaction in which the substrate is con- verted to a colored precipitate directly on the membrane, essen tially coloring the band on which the primary antibody has bound While not as sensitive as other methods colorimetric detection is fast and simple, and requires no special facilities. Chemiluminescent detection combines characteristics of both radioactive and chromogenic detection. Again, an enzyme label is used(commonly HRP, but there are systems for use with AP as well), but in this case the reaction produces light rather than a colored product as a result of reaction. The light is usually cap tured on X-ray film, just like a radioactive blot. Specialized imaging equipment for chemiluminescent blots is also available Chemiluminescent detection is very sensitive, and the blots are easily stripped for subsequent reprobing There are significant differences in the various available chemi luminescent detection systems. The most widely used are the luminol-based HRP systems. These typically emit usable signals for an hour or two. There are also newer, higher-sensitivity HRP- based systems that emit light for more than 24 hours; however, these substrates are more expensive and require even more careful optimization than the luminol-based systems. AP-based hemiluminescent systems are also available. They are not also emit light for extended periods. Those systems produc- ing extended light output have the advantage that several ex posures can be taken from the same blot With the availability of fluorescence-scanning instruments, new methods for detection have come into use. It may seem at first glance that a secondary antibody could simply be coupled to a flu orescent molecule and the detection performed directly. Although this is possible, this method is not sufficiently sensitive for most purposes. The approach usually taken uses an enzyme-coupled 376 Riiswashed and exposed to film for hours or days. Radioactive blots can more quickly be detected using storage phosphor plates instead of film; the plates are read on a specialized scanning instru￾ment. Detailed discussions about the features and benefits of detection by film and scanners are included in Chapter 14, Nucleic Acid Hybridization. Enzymatic reactions are used in a number of different systems to indicate the presence of bound antibody. The simplest type of enzymatic detection is chromogenic. Here the secondary reagent is conjugated to an enzyme, either horseradish peroxidase (HRP) or alkaline phosphatase (AP). After incubation with the sec￾ondary reagent and washing, the blot is incubated with a substrate. The enzyme catalyzes a reaction in which the substrate is con￾verted to a colored precipitate directly on the membrane, essen￾tially coloring the band on which the primary antibody has bound. While not as sensitive as other methods, colorimetric detection is fast and simple, and requires no special facilities. Chemiluminescent detection combines characteristics of both radioactive and chromogenic detection. Again, an enzyme label is used (commonly HRP, but there are systems for use with AP as well), but in this case the reaction produces light rather than a colored product as a result of reaction. The light is usually cap￾tured on X-ray film, just like a radioactive blot. Specialized imaging equipment for chemiluminescent blots is also available. Chemiluminescent detection is very sensitive, and the blots are easily stripped for subsequent reprobing. There are significant differences in the various available chemi￾luminescent detection systems. The most widely used are the luminol-based HRP systems. These typically emit usable signals for an hour or two. There are also newer, higher-sensitivity HRP￾based systems that emit light for more than 24 hours; however, these substrates are more expensive and require even more careful optimization than the luminol-based systems. AP-based chemiluminescent systems are also available. They are not widely used in Western blotting, but they are highly sensitive and also emit light for extended periods. Those systems produc￾ing extended light output have the advantage that several ex￾posures can be taken from the same blot. With the availability of fluorescence-scanning instruments, new methods for detection have come into use. It may seem at first glance that a secondary antibody could simply be coupled to a flu￾orescent molecule and the detection performed directly.Although this is possible, this method is not sufficiently sensitive for most purposes. The approach usually taken uses an enzyme-coupled 376 Riis