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Detection and Imaging

There are a variety of detection systems to choose from based on chemiluminescent, fluorescent, or radioisotopic reagents.

Enzyme-based chemiluminescence and direct fluorescence are the most popular detection methods used in Western blotting.

Chemiluminescence is used for routine protein identification and highly sensitive detection of low abundance proteins.

Fluorescence detection provides accurate quantitative WB data due to its signal stability and broad linear range. On top of this, fluorescence detection enables multiplexing applications.

Typhoon high performance scanners offer the versatility to use filmless phosphor imaging and multiplex fluorescence imaging.

Amersham Imager 600 series provides excellent image quality for a broad range of applications, including chemiluminescence, multiplexing fluorescence, and densitometry.

Chemiluminescence Fluorescence Storage Phosphor

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.

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 the method of choice for quantitative WB due to its signal stability, broad linear dynamic range, and the fact that it allows for multiplexed detection.

Fluorescence detection is a direct method where the secondary antibody is conjugated to a fluorophore, thus avoiding the need for additional detection reagents. During fluorescence detection, light is emitted by the fluorophore after excitation via light of a specific wavelength. A laser scanner or a CCD camera-based imager can be used to collect and convert the emitted light to a digitized signal for image display and analysis.

Amersham ECL Plex is a fluorescence labeling kit for quantitative, multiplex detection of two proteins simultaneously.

Radioactive systems in combination with phosphorimaging screens are still used in Southern and Northern blotting because of their sensitivity and speed.

Storage phosphor screens or phosphorimaging plates (IP) are reusable (except for tritium screens) and can capture two-dimensional images of radiolabels.

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.

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.

 

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