There are six basic steps in the Western blotting procedures with the Phototope®-HRP Western Blot Detection System.
7074 Anti-rabbit IgG, HRP-linked Antibody: Store at -20°C.7727 Biotinylated Protein Ladder Detection Pack: Store at -20°C.7003 20X LumiGLO® Reagent and 20X Peroxide: Store at 4°C.
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.
Chemiluminescence systems have emerged as the best all-around method for western blot detection. They eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives, and because results are generated on film, it is possible to record and store data permanently. Blots detected with chemiluminescent methods are easily stripped for subsequent reprobing with additional antibodies. HRP-conjugated secondary antibodies are utilized in conjunction with specific chemiluminescent substrates to generate the light signal. HRP conjugates have a very high turnover rate, yielding good sensitivity with short reaction times.
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