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Phototope®-HRP Western Blot Detection System, Anti-mouse IgG, HRP-linked Antibody #7072
System Includes
- Anti-mouse IgG, HRP-linked Antibody #7076
- Anti-biotin, HRP-linked Antibody #7075
- Biotinylated Protein Ladder Detection #7727
- LumiGLO® Reagent and Peroxide #7003
Description
The Phototope®-HRP Western Blot Detection System is designed for the chemiluminescent detection of proteins in standard Western blotting applications. Proteins and biotinylated molecular weight markers (provided) are separated by SDS-PAGE and transferred onto membrane. Following incubation with your primary anti-serum, horseradish peroxidase (HRP) linked secondary antibody and HRP-linked anti-biotin antibody are bound and then allowed to react with LumiGLO® reagent. The light emitted by destabilized LumiGLO reagent is subsequently captured on X-ray film.
System Includes
- Anti-mouse IgG, HRP-linked Antibody #7076
- Anti-biotin, HRP-linked Antibody #7075
- Biotinylated Protein Ladder Detection Pack #7727
- LumiGLO® Reagent and Peroxide #7003
Western Blotting
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.
Applications
This product has been optimized for use in chemiluminescent Western blotting applications.
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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