When doing a Western blot procedure, the macromolecules are first separated using gel electrophoresis. The separated macromolecules are transferred onto a second matrix (nitrocellulose or polyvinylidene difluoride (PVDF) membrane) and the membrane is blocked to prevent the nonspecific binding of antibodies to its surface. The transferred protein is then complexed with an enzyme-labeled antibody (probe) and an appropriate substrate is added to produce a detectable product.
Most researchers previously used radioisotopes for this purpose, but since they are expensive, have a shorter shelf-life, and does not offer any improvement in signal: noise ratio, alternative labels such as enzymes and fluorophores took over their place. In addition, radioisotopes require special handling and disposal, something that most researchers can do without.
Protein Detection Methods: Chromogenic vs. Chemiluminescent Detection
Enzymatic labels such as alkaline phosphatase (AP) and horseradish peroxidase (HRP) are commonly used by researchers for protein detection since they are extremely sensitive, especially when optimized with an appropriate chromogenic and/or chemiluminescent substrate. So, which of these two detection systems is better and which one should you use? Here is a rundown of all the advantages and disadvantages of each method.
Chemiluminescent Detection
Chemiluminescent protein detection currently reigns as the method of choice for most laboratory researchers due to a number of reasons. Among these are the following:
- It allows multiple exposures thereby providing researchers with the best possible image.
- Since the detection reagents can be removed and the entire blot reprobed, it allows researchers to visualize another protein or optimize the detection of the first protein with relative ease.
- It allows detection and quantitation for a large range of protein concentrations.
- It provides the greatest sensitivity as compared to other available detection methods.
In this method, the chemiluminescent substrate produces light when combined with the enzyme. The resulting light output can be captured using film, a CCD camera or a phosphorimager that is specifically designed for this purpose. The intensity of the signal determines the abundance of the antigen on the membrane.
However, please take note that the signal will start to weaken and eventually cease once the substrate is exhausted or the enzyme loses activity. To make sure you have ample time to detect and document your results, you should make sure that you have a properly optimized assay. Use proper antibody dilutions and sufficient substrate and you can produce a stable output of light for several hours.
Chromogenic Protein Detection
Researchers have been using chromogenic or precipitating substrates for a number of years primarily because they provide the simplest and most cost-effective method of detecting your protein of interest. And unlike the other method, it requires no special equipment for processing or visualizing.
Under this method, these substrates are converted into insoluble, colored products that precipitate onto the membrane once they come in contact with the appropriate enzyme. The colored precipitate cannot be easily stripped off so you need to allow the reaction to proceed until color development has reached a satisfactory level. Chromogenic blotting substrates are available in a wide variety of specifications and formats so you can choose the appropriate substrate depending on the enzyme label, desired sensitivity and form of signal and/or method of detection needed.
Unfortunately, this method is not as sensitive as the other protein detection methods available so you should not use it when detecting proteins of low abundance. In addition, the chromogenic substrates have a tendency to fade as the blot dries or when it is stored. To overcome this limitation, you may choose to photocopy or scan the blot so you will have a permanent replica of your results.
So, which of these two detection systems is better and which one should you use? Well, if you are working with applications where protein abundance is high, you should choose the chromogenic method since it will provide a stable signal and will not produce any false negative results (ghost bands) such as those that occur with chemiluminescent substrates.
Image Source : Luis Romero
