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Western blot aims to identify specific proteins within a complex mixture. The western blot technique requires samples to be resolved based on size through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), following which they are transferred to and immobilized on a membrane before antibody-based detection.
Before running a western blot, it is extremely important to research the target protein thoroughly. To learn more about the procedure, refer to our western blot protocol.
You can watch our on-demand western blot webinar for more information on the western blot procedure.
There are several different choices of readout when western blotting. Each has advantages and disadvantages, which depend on your needs and equipment available in your lab.
Figure 1. Schematic representation of colorimetric western blot detection. The left panel demonstrates indirect detection while the right panel shows direct detection.
Colorimetric detection relies on the generation of a colored product that becomes deposited on the western blot, which is formed following the conversion of a chromogenic blotting substrate by an appropriate enzyme. The limited sensitivity of chromogenic substrates can make it difficult to optimize them for detecting proteins of low abundance, although the chromogenic reaction can be allowed to develop for several hours (or even overnight) to allow the background signal to develop simultaneously.
However, colorimetric substrates are perfect for the detection of abundant proteins since the reaction can be monitored visually and allowed to progress until there is adequate color development before being stopped. No specialized equipment is required for visualization of the colored precipitate, and the produced signal is highly stable.
Figure 2. Schematic representation of fluorescent western blot detection.
Fluorometric detection requires the use of an antibody which has been labeled with a fluorophore. A light source is used to excite the fluorophore, which then produces a transient light emission as it returns to its ground state. The light is emitted at a higher wavelength than that which was used for excitation and is detected with a specialized reader.
Figure 3. Schematic representation of chemiluminescent western blot detection.
Chemiluminescence occurs when a substrate is catalyzed by an enzyme and produces light as a byproduct of the reaction. The limiting reagent in the reaction is the substrate – as this is exhausted, the light production decreases and eventually stops. However, a well-optimized procedure should produce a stable light output for several hours, allowing consistent and sensitive protein detection.
For more information, check out our library of western blot resources.