Detection and Imaging
There are a variety of detection systems to choose from based on chemiluminescent, ﬂuorescent, chemiﬂuorescent, radioisotopic, or chromogenic reagents.
Radioisotopic and chromogenic reagents have been widely used for many years, but safety issues such as waste handling and disposal procedures have led to a decline in their popularity.
Enzyme-based chemiluminescence and chemiﬂuorescence detection, as well as direct ﬂuorescence, have been extensively developed and are now the most popular detection methods due to their improved sensitivity and wider linear dynamic range.
Chemiluminescence and chemiﬂuorescence require the addition of a substrate that generates a light signal when it reacts with enzyme conjugated to a secondary antibody. Direct fluorescence detection, on the other hand, requires no additional reagents.
Since the early 1990s, GE Healthcare has developed chemiluminescence detection systems that are now among the most widely used for Western blotting applications. Choose an Amersham ECL system that suits your needs, depending on the purpose of your experiment.
Amersham ECL provides fast and reproducible results with minimal optimization, and is therefore excellent for routine detection and analysis.
Amersham ECL Prime provides outstanding signal intensity and sensitivity, achieving high quality results with highly diluted primary and secondary antibodies. Stable signal intensity makes it easier to process several blots in the same run.
Chemiluminescence detection is based on the reaction between horseradish peroxidase (HRP)-labeled antibodies and luminol substrate. In the presence of HRP, hydrogen peroxide catalyzes the oxidation of luminol, a reaction that results in the emission of light. The light signal can then be detected on X-ray ﬁlm or by digital imaging with a charge-coupled device (CCD) camera-based imager.
Fluorescence detection is a direct method where the secondary antibody is conjugated to a ﬂuorophore, thus avoiding the need for additional detection reagents. The system is highly sensitive, delivers a broad linear dynamic range, allows multiplexed detection, and is well adapted to quantitative Western blotting.
During ﬂuorescence detection, light is emitted by the ﬂuorophore after excitation via light of a speciﬁc wavelength. A photomultiplier tube (PMT) or a CCD camera-based imager can be used to collect and convert the emitted light to an electrical signal. The electrical signal is then digitized for image display and analysis.
We offer fluorescent-labeling reagents, fluorescently labeled antibodies, as well as convenient kits such as Amersham ECL Plex for quantitative, multiplex detection of two proteins simultaneously.
In addition to direct detection via ﬂuorophores, indirect chemiﬂuorescence systems are also available. Chemiﬂuorescence is based on AP-conjugated antibodies, which react with a ﬂuorogenic substrate to generate a stable ﬂuorophore. The advantage of AP is that its reaction rate remains linear over time; sensitivity may thus be increased by prolonging incubation time with substrate.
Enhanced chemifluorescence (ECF) has a similar limit of detection as enhanced chemiluminescence (ECL), but has the advantage that the signal from the stable ﬂuorophore can be detected on multiple occasions.
Autoradiographic films are photographic films that can capture the images created by electromagnetic radiation, or particles emitted by radioisotopes. They can be used to detect radiation from radioactively-labeled probes and also the light generated by, for example, chemiluminescence methods.
The use of autoradiography as a detection method in Western blotting applications has greatly diminished due to cost and safety issues, such as the need to work in a radioactive environment, extensive waste handling, etc. Despite these issues, autoradiography is very sensitive and is compatible with most of the imagers available from GE Healthcare. Autoradiographic detection is either performed using an X-ray ﬁlm or a storage phosphor screen.
GE Healthcare offers a range of imagers and image analysis software for several techniques including chemifluorescence, fluorescence, filmless phosphor imaging, and colorimetry.
Typhoon high performance scanners offer the versatility to use filmless phosphor imaging, multiplex fluorescence imaging, and chemifluorescence.
ImageQuant CCD-based imagers cover a range of monochromatic imaging applications including chemiluminescence imaging, monochromatic fluorescence, and colorimetry.
Filmless Phosphor Imaging
Some scanners, such as Typhoon, are able to acquire radioactive signals using a reusable storage phosphor screen, which captures latent images produced by ionizing radiation, such as emissions from radioisotopes.
When stimulated with a laser, light is emitted from the storage phosphor screen in proportion to the amount of radioactivity in the sample and can be detected. Storage phosphor screens avoid the need for autoradiography film, development chemicals, and a darkroom. They are very sensitive and may require only one tenth of the exposure time required by autoradiography film.