Western blotting is an old, but widely used biotechnology technique used to detect proteins in a sample. Given its commonality and importance in scientific research, here is everything you need to know about western blots.
What exactly is western blotting? It is a technique that allows scientists to identify and analyze a protein of interest within a sample of mixed proteins. This technique is heavily based on the transfer or blotting of proteins to a solid membrane, so that proteins can be detected by antibodies. Once the protein of interest is detected, it can be further analyzed.
- Denature and Separate Proteins
This first step can be accomplished in many ways, but is most commonly done by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS is a detergent that will denature proteins or break them down by unfolding them, and giving them an overall negative charge. Once the proteins have been denatured, they can be separated by gel electrophoresis. The samples can be loaded onto a polyacrylamide gel, and the SDS denatured proteins will be separated by size as they travel towards the positive electrode during electrophoresis.
- Transfer of Proteins to Membrane
As mentioned before, the transfer of proteins from the gel to a membrane is an important aspect of this technique. After electrophoresis, protein transfer can take place. This can be accomplished in many ways, but traditionally is done through a wet transfer. Filter paper, a gel, and a nylon membrane are put into a stack where each piece is put in close contact with each other. The order of the stack from the bottom to the top is as follows: filter paper, gel, nylon membrane, and another piece of filter paper. The bottom of the stack will be wet and is drier towards the top. For the transfer to occur, the nylon membrane must be stacked directly on top of the gel. The transfer of proteins occurs because of diffusion, as the stack enables the buffer to travel towards the top where it is drier. A nylon membrane is used because it is a very stable material, making it a great surface to manipulate proteins on.
- Visualization of Protein
Depending on what type of protein analysis is desired, the visualization technique used will be different. If looking only at the total amount of protein that was transferred, then a simple protein dye can be used to stain the membrane and visualize the amount of protein present. If you are looking to detect one specific protein from a sample that contained a mixture of proteins, an immunological detection method needs to be used. This method utilizes antibodies that will be able to detect the protein of interest. After the transfer, the membrane will be placed in a blocking buffer, so that the antibody will only bind to the protein on the membrane. Next, the membrane is exposed to a buffer with the antibody of the protein of interest. The antibody will bind to the protein of interest, then the membrane is washed to remove all unbound antibodies. A secondary antibody that is covalently linked to an enzyme, like horseradish peroxidase, is exposed to the membrane. This secondary antibody will bind to the primary antibody-protein complex, and the membrane is washed to remove all unbound secondary antibody. Lastly, the membrane will be exposed to a peroxidase substrate, so that the enzyme will go through a reaction and produce a chromogenic product. In other words, the protein is now visible on the membrane as a band of color, due to the enzymatic reaction.
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