Western Blot Detection Reagents

Chemiluminescent western blot detection

Chemiluminescent substrates are popular because they offer several advantages over other detection methods. These advantages have allowed chemiluminescence to become the detection method of choice in most protein laboratories. Using chemiluminescence allows multiple exposures to be made in order to obtain the best image. The detection reagents can be removed and the entire blot reprobed to visualize another protein or to optimize detection of the first protein. A large linear response range allows detection and quantitation for a large range of protein concentrations. Most importantly, chemiluminescence yields the greatest sensitivity of any available detection method.

Chemiluminescent substrates for horseradish peroxidase (HRP) are majority two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. Chemiluminescent substrates for alkaline phosphatase are also readily available. Click here for information on chemiluminescent western blot detection can be found here. 

Fluorescent western blot detection

Fluorescent reagents are growing in popularity for western blotting because they offer increased time savings over chemiluminescent detection and reduced chemical waste compared to both chemiluminescent or chromogenic detection systems. While the detection limits are still not as low as chemiluminescent detection, fluorescent detection has the unique advantage of allowing multiple targets to be assayed for on the same blot at the same time without the need to strip and reprobe.

The growing demand for multiplex western blotting has driven the development of many new fluorescent dyes (such as Alexa Fluor Plus conjugates). These new fluorophores are brighter and more photo stable than the traditional fluorescein and rhodamine molecules traditionally used and comprise a broader range of non-overlapping spectra. Together with the advances in the digital imaging equipment these new fluorophores enable extremely powerful analyses in western blotting. Click here for information on fluorescent western blot detection can be found here.

Chromogenic western blot detection

Chromogenic or precipitating substrates have been used widely for many years and offer the simplest and most cost-effective method of western blot detection. When these substrates come in contact with the appropriate enzyme (e.g. Alkaline phosphatase- AP or horseradish peroxidase-HRP), they are converted to insoluble, colored products that precipitate onto the membrane. The resulting colored band or spot requires no special equipment for processing or visualizing. Chromogenic blotting substrates are available in a variety of specifications and formats, producing a range of colored precipitates. The appropriate substrate choice depends on the enzyme label and desired sensitivity. Similar to developing film, the blot is incubated in substrate until the desired amount of development is achieved. In contrast to chemiluminescent western blotting, the colored precipitate formed by chromogenic substrates cannot be easily stripped off to facilitate re-probing procedures. Therefore, it is important to allow the reaction to proceed until color development is satisfactory and then stop the reaction.

The low sensitivity of chromogenic substrates makes it difficult to optimize for detecting proteins of low abundance. Although the reaction can be allowed to develop for several hours or even overnight, this also allows background signal to continue to develop. Where chromogenic substrates fail in terms of sensitivity, they are ideal for applications where protein abundance is high. Click here for information on chromogenic western blot detection can be found here.