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- Analytical tools
Phototope-HRP Western Blot Detection System, Anti-mouse IgG, HRP-linked Antibody #7072
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.
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.
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.
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.