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20X LumiGLO® Reagent and 20X Peroxide #7003
Description
LumiGLO®* chemiluminescent substrate is a luminol-based system designed for use with our Phototope®-HRP detection assays utilizing peroxidase-labeled antibodies immobilized on membranes. In the presence of hydrogen peroxide, horseradish peroxidase (HRP) converts luminol to an excited intermediate dianion. This dianion emits light on return to its ground state. Light emission is maximal immediately after exposure of the substrate to HRP and continues for 0.5-1 hour. Light can be captured on X-ray film, typically by exposure for a few seconds. Maximum sensitivity can be obtained by longer exposure.*Avoid repeated exposure to skin (see enclosed Material Safety Data Sheet or refer to our website for further information).
Western Blotting
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Method Overview
There are six basic steps in the Western blotting procedures with the Phototope®-HRP Western Blot Detection System.
- Polyacrylamide Gel Electrophoresis of Proteins: Separate the protein samples and molecular weight standards by polyacrylamide gel electrophoresis.
- Transfer: Transfer the protein to membrane by standard electroblotting.
- Block Membrane: Block to saturate nonspecific binding sites on the membrane.
- 1° Antibody: Incubate the membrane with the primary antibody.
- 2° Antibody: Incubate the membrane with HRP-linked anti-rabbit IgG and HRP-linked anti-biotin antibodies.
- Chemiluminescent Detection: Add LumiGLO® reagent and capture the emitted light on X-ray film.
Background
Chemiluminescent detection systems have emerged as the best all-around method for detection of Western blots. They eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives. Because results are generated on film, it is possible to record and store data permanently, and blots detected with chemiluminescent methods are easily stripped for subsequent reprobing with additional antibodies. Horseradish peroxidase (HRP) conjugated secondary antibodies are utilized in conjunction with specific chemiluminescent substrates to generate the light signal. Horseradish peroxidase-antibody conjugates have a very high turnover rate, giving good sensitivity with short reaction times.
Application References
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Companion Products
- 7071 Phototope®-HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
- 7072 Phototope®-HRP Western Blot Detection System, Anti-mouse IgG, HRP-linked Antibody
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 7075 Anti-biotin, HRP-linked Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 7077 Anti-rat IgG, HRP-linked Antibody
