Difference Between Western Blotting and Far Western Blotting
Blotting techniques have enabled scientific advancement, especially in the medical field, for decades now. Building on the foundation of the northern and southern blots, W Neal Burnette developed the western blot to isolate and identify target proteins within a mix of cell materials.
Later, scientists built upon the western blot to create the far-western blot. Rather than studying proteins individually, the far-western blot turns its attention to the interactions between proteins. This has a variety of applications both distinct from and related to those of the western blot. Let’s take a look at each.
The western blot method relies on cell lysis and gel electrophoresis to separate out proteins of interest. This enables researchers to study specific proteins in isolation rather than within a mix of cell tissues, organelles and other proteins, which would prove difficult to impossible to utilize efficiently.
How Does Western Blotting Work?
To isolate proteins, scientists use a fairly straightforward method:
- Using various chemicals, they break open cell membranes – a process known as lysis – without destroying the contents within. Preventing the degradation, breaking or contamination of proteins is a crucial step here.
- The proteins are then placed in a gel with pores of different sizes in it. Running an electrical current through the gel causes the proteins to move at different rates and separate out by size.
- Scientists then move the proteins of interest to a new membrane using a transfer process: wet, semi-dry or rapid semi-dry. The best method depends on the protein.
- They then use primary antibodies to bind to the protein, then attach secondary antibodies tagged in such a way as to make them visible to the primary antibodies. After each application, the sample is washed.
- Now it’s time to collect the data by transferring images to a software program for study.
What Is It Used For?
The western blot can be used to discover the size of a protein, as well as to measure protein expression. It is employed in molecular biology and immunology to determine the basic characteristics of proteins and the effects of protein modifications. It is frequently used to detect antibodies in testing for disease and is instrumental in some tests for doping.
Understanding how proteins interact with one another is just as important as isolating the proteins themselves. This allows researchers to understand basic bodily functions, how drugs work within the human body, and more.
How Does Far-Western Blotting Work?
Because there are multiple types of proteins involved, they are separated into two main categories: prey proteins and bait proteins. The latter is a known protein, while the former is the unknown focus of the study. Ensuring they interact effectively and reproducibly is the main challenge of far-western blotting. The method works like so:
- Separate out bait and prey proteins. This usually relies on the western blot method to isolate each one.
- Tag bait proteins so they are identifiable in the experiment.
- Both bait and prey proteins are added to a membrane or to gel, and researchers use a known enzyme to detect the interactions between them.
- Scientists then capture images of the process, as they do with the Western blot, and transfer them to software to examine the data.
What Is It Used For?
The far-western blot has many applications, but one of the most common is the detection of receptor-ligand interactions. These are involved in many metabolic, neurological and cellular signal pathways, and lead to important physiological responses in the body. Understanding them is critical to addressing a variety of pharmacological and health topics.
Also, the far-western blot is making major contributions to protein-protein interaction libraries, continuously building our medical and scientific knowledge.
In summation, the far-western blot could not exist without the western blot, whereas the western blot would not be nearly as useful without its far-western companion. Continuing to develop the efficacy and reproducibility of both will be key in the coming decades.