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Product 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.


Product Usage Information

Protocols

Product Usage Information

There are six basic steps in the Western blotting procedures with the Phototope®-HRP Western Blot Detection System.

  1. Polyacrylamide Gel Electrophoresis of Proteins: Separate the protein samples and molecular weight standards by polyacrylamide gel electrophoresis.
  2. Transfer: Transfer the protein to membrane by standard electroblotting.
  3. Block Membrane: Block to saturate nonspecific binding sites on the membrane.
  4. 1° Antibody: Incubate the membrane with the primary antibody.
  5. 2° Antibody: Incubate the membrane with HRP-linked anti-rabbit IgG and HRP-linked anti-biotin antibodies.
  6. Chemiluminescent Detection: Add LumiGLO® reagent and capture the emitted light on X-ray film.

Storage: 7076 Anti-mouse 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.

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.


Data Sheets & Documentation


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.