How to identify proteins using western blot reagents?
Basically, Western blotting is a technique used in the process of protein identification. In order to accurately identify the proteins present in a particular sample, individual protein bands are initially separated and sorted according to their physical characteristics using gel electrophoresis. After this initial step, the separated protein bands are transferred to a nitrocellulose or PVDF membrane and are then treated with specific antibodies and appropriate Western blot reagents to complete the process.
Prior to treatment with antibodies, the membrane is incubated with a blocking agent. For more details on blocking agents, read our “Which Blocking Agent for Western Blotting” blog.
When using conventional or indirect detection methods of Western blotting, the proteins are initially incubated with a solution of primary antibody that recognizes the protein(s) of interest. The bound primary antibodies are then washed away and are treated with a secondary antibody which binds to the constant domain of the primary antibody. This process intensifies the signal intensity and provides a better resolution for more accurate protein identification.
The proteins are “visualized” by using a specific label conjugated to the secondary antibodies. These labels include fluorescent probes or enzymes, such as horseradish peroxidise (HRP) or alkaline peroxidise (AP). The enzymes react with specific substrates to either produce a colored precipitate on the membrane or light (chemiluminescence). The light produced from this reaction can be captured using film, a CCD camera, a phosphorimager or a fluorescence imaging system.
After washing away unused probes, the blots are analyzed using the following methods:
Colorimetric detection. This method uses spectorophotometry or densitometry to analyze the quantity of proteins. However, since this method gives low resolution when there is insufficient amount of proteins available, it should only be used when there is abundant protein in the blot.
Fluorescent detection. In this method, the western blot reagents are exposed to light and the resulting blots are analyzed using a photo sensor or a CCD camera. Another alternative to detection is the use of fluorescence-labeled probes.
Chemiluminescent detection. This method is considered to be far more superior as compared to fluorescent detection since it is more sensitive, allows protein identification and detection over a wider range of concentration and produces fast results.
While western blotting may be more time-consuming as compared to other immunosorbent assays such as ELISA, it allows for the detection of several types of proteins and makes semi-quantification possible.