Chemiluminescence Western blots are probed with a primary antibody against the target protein, followed by a secondary antibody labeled with HRP (horseradish peroxidase) enzyme. A chemiluminescence substrate for the HRP enzyme is carefully applied to the blot, and light is emitted when the HRP enzyme modifies the substrate. Photographic film or an imaging system using a digital CCD camera captures the emitted light as an image. Most importantly, chemiluminescence yields the greatest sensitivity of any available detection method.
The most popular Western blot substrates are luminol-based and produce a chemiluminescent signal. Chemiluminescence is a chemical reaction that produces energy released in the form of light. In the presence of horseradish peroxidase (HRP) and a peroxide buffer, luminol oxidizes and forms an excited state product called 3-aminophthalate that emits light at 425 nm. The emission ends when 3-aminophthalate decays to the ground state. Light emission occurs only during the enzyme-substrate reaction and, therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases. In contrast, colorimetric substrates, such as DAB, produce precipitate that remains visible on the membrane even after the reaction has terminated. Several varieties Chemiluminescent Substrates for HRP are available that provide for different levels of sensitivity for chemiluminescence Western Blot.
Exposure to light imprints an image on X-ray film. The amount of light that reaches the film determines the image, with brighter, more intense light producing a stronger image. In Western blotting, this light comes from labeling proteins with chemiluminescent or radioactive signals. In chemiluminescent labeling, horseradish peroxidase (HRP)-conjugated secondary antibodies react with enhanced chemiluminescence (ECL) reagents to emit light. The intensity of imprint on an X-ray film is proportional to the signal. In turn, this signal relates to the amount of protein present on the Western blot membrane, which allows quantitation.
CCD camera imaging is suitable for chemiluminescence and fluorescence detection. This technique uses a light source to illuminate the membrane, when needed, or excite fluorophores at specific wavelengths. Lenses collect the resulting light from the imaging field, focusing it on a two-dimensional CCD array. The resulting digital image is essentially a pattern of charge proportional to the light from the chemiluminescence reaction or fluorescent dyes. The intensity of the image is proportional to the amount of light produced, indicating protein quantity.
In addition, Sino Biological offers a range of secondary antibodies conjugated with HRP enzymes. These secondary antibodies can be used in chemiluminescence Western Blot detection.
|X-ray film||CCD camera|
|Strengths||• High sensitivity
• Flexible exposure times for detecting weak signals
|• Broad linear dynamic range
• High sensitivity
• Quantitative imaging
• Digital image produced
|Limitations||• Narrow linear dynamic range
• Requires a dark room
|• Requires CCD cooling measures|