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.
The HRP Western Blot Detection System is designed for the chemiluminescent detection of proteins in standard Western blot 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 antibiotin 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.
This product has been optimized for use in chemiluminescent Western blot applications.
There are six basic steps in the Western blot procedures with the 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 degrees Antibody: Incubate the membrane with the primary antibody.
5. 2 degrees 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.
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.
Sensitivity: Detection of subpicogram amounts of protein is routine with good primary antisera.
Assay Time: Less than 1 hour is required for the entire detection procedure. Exposure times are seconds to minutes
Multiple Exposures: Light is emitted at a constant rate for several minutes, so you can perform multiple exposures to optimize signal intensity. Re-exposure at a future date is achieved by simply adding more reagent.
Stability: A permanent hard-copy record is generated that will not fade or disintegrate over time.
Quantitative: X-ray films can be scanned to quantitate band intensities.
1. Kits are available for rabbit and mouse primary antisera.
2. Simultaneous detection of biotinylated molecular weight standards