In this article, I briefly describe western blotting and its applications.
Western blotting
The technique of identifying a specific protein in a complex mixture of proteins is known as western blotting. This was developed by Towbin, et al. in 1979. It is also called immunoblotting as an antibody is specifically used to detect its antigen.
Procedure
Gel electrophoresis
The procedure begins with extracting protein from various samples, such as tissue or cells. Protein samples are often denatured by boiling before electrophoresis. Then, a mixture of proteins is separated by electrophoresis on an SDS-polyacrylamide gel (SDS-PAGE). It is a slab gel infused with sodium dodecyl sulfate (SDS), a dissociating agent (figure 1). SDS is generally used as a buffer (and in the gel) to give all proteins a uniform negative charge since proteins can be positively, negatively, or neutrally charged.
Proteins are separated using polyacrylamide gel electrophoresis to characterize individual proteins in a complex sample or examine multiple proteins within a single sample (PAGE). When combined with Western blotting, PAGE is a powerful analytical tool providing information on the mass, charge, purity, or presence of a protein. Many forms of PAGE provide various information about the protein (s).
Proteins are treated with strong reducing agents to remove the secondary and tertiary structures. SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains the polypeptides in a denatured state. Thus, it allows the separation of proteins by their molecular weight. SDS is negatively charged and coats the sampled proteins, which move to the positively charged electrode through the acrylamide gel. Smaller proteins migrate faster through this mesh. Thus, the proteins are separated according to size (usually measured in kilodaltons, kDa).
The resolution of the gel depends upon the concentration of acrylamide
The concentration of acrylamide determines the resolution of the gel. The higher acrylamide concentration defines the better resolution of lower molecular weight proteins. The lower acrylamide concentration defines the better resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots. The gel consists of wells and samples are loaded into the wells in the gel. When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.
Transfer of proteins to a membrane
Proteins are transferred to a nitrocellulose or polyvinylidene difluoride membrane (PVDF) to make them accessible for antibody detection. The various methods of protein transfer can be categorized as diffusion transfer, capillary transfer, heat-accelerated convectional transfer, vacuum blotting transfer, and electrophoretic transfer.
In capillary transfer, the membrane is placed on top of the gel, and a stack of filter papers is placed on top. The entire stack is placed in a buffer solution. The paper moves up by capillary action bringing the proteins with it. However, capillary transfer is a time-consuming method.
The electrophoretic transfer method is generally used for its speed and transfer efficiency. This method involves placing a protein-containing polyacrylamide gel in direct contact with a piece of nitrocellulose or other suitable, protein-binding support. Then, this is sandwiched between two electrodes submerged in a conducting solution. On application of an electric field, the proteins move out of the polyacrylamide gel onto the surface of the membrane. The proteins are tightly attached to the membrane surface (figure 1). This results in a membrane with a copy of the protein pattern, which was originally in the polyacrylamide gel. The membrane is stained with Coomassie Brilliant Blue to check the uniformity and overall effectiveness of the transfer of protein from the gel to the membrane.
Blocking of the membrane
The membrane surface is blocked after the transfer of proteins from the gel. This is done to prevent non-specific binding of the detection antibodies during the coming steps. The blocking is done by placing the membrane in a dilute solution of protein. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no space for its attachment other than on the binding sites of the specific target protein. This leads to clearer results and eliminates false positives.
Detection
In the detection process, the membrane is tested for the target protein using a modified antibody that is attached to a reporter enzyme. When this enzyme interacts with a specific substrate, it catalyzes a colorimetric reaction, resulting in a visible color change.
Exposure to the primary antibody
Western blotting usually involves probing the blocked membrane with a primary antibody that targets a specific protein or epitope, such as an SH2 domain or phosphorylated tyrosine. The selection of the primary antibody depends on the antigen being detected and the available antibodies for that antigen.
Following the blocking step, the membrane is incubated with a diluted primary antibody solution, which typically contains buffered saline, a small amount of detergent, and occasionally powdered milk or BSA. This incubation, done under gentle agitation, can last from 30 minutes to overnight. If incubating overnight, it is crucial to do so at 4°C to avoid contamination and degradation of the protein, especially phosphorous groups. Agitation helps ensure even distribution of the antibody across the membrane and prevents uneven binding.
Exposure to the secondary antibody
After washing the membrane to eliminate any unbound primary antibody, it is treated with a secondary antibody that targets a part of the primary antibody specific to the species. Various labeled secondary detection reagents can be utilized for Western blotting. Typically, the secondary antibody is conjugated with biotin or a reporter enzyme like alkaline phosphatase or horseradish peroxidase. This conjugation allows multiple secondary antibodies to bind to each primary antibody, thereby amplifying the signal.
Means of detection
Enzymatic labels are frequently utilized in Western blotting. Although these labels involve additional steps, they can be highly sensitive when optimized with the right substrate. The enzymes most commonly used for protein detection are alkaline phosphatase (AP) and horseradish peroxidase (HRP). HRP is the most preferred one due to its high activity rate, good stability, low cost, and the wide availability of its substrates. After the enzyme-antibody conjugate binds, a chromogenic substrate is added, resulting in a highly colored, insoluble product that forms a visible band at the site of the target antigen.
The second type of secondary antibody detection is a fluorescent detection method. It uses a near-infrared (NIR) fluorophore-linked antibody. This technique offers a more precise and accurate measurement of the signal differences produced by labeled antibodies bound to proteins on a Western blot. Unlike chemiluminescence, where light is measured dynamically, fluorescent detection measures the signal in a static state, allowing for accurate quantification of the proteins on the membrane. Additionally, the use of fluorophore-conjugated antibodies (figure 2) simplifies the process as it eliminates the need for a substrate development step. However, this method requires specialized equipment to detect and record the fluorescent signal due to the necessity of an excitation light source.
A radioactive label can also be used instead of an enzyme and coupled to the secondary antibody. When the protein of interest is bound by a radioactive antibody, its position on the blot can be identified by exposing the membrane to a sheet of X-ray film. The process is known as autoradiography.
Conclusion
Western blotting is the technique of identifying a specific protein in a complex mixture of proteins. In this process, an antibody is specifically used to detect its antigen. The procedure begins with extracting protein from various samples, such as tissue or cells. Protein samples are often denatured by boiling before electrophoresis. Then, a mixture of proteins is separated by electrophoresis on an SDS-polyacrylamide gel (SDS-PAGE). SDS is negatively charged and it coats the sampled proteins, which move to the positively charged electrode through the acrylamide gel. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilodaltons, kDa).
The proteins are transferred from a gel to a nitrocellulose membrane to make them accessible for antibody detection. The membrane surface is blocked after the transfer of proteins from the gel. This is done to prevent non-specific binding of the detection antibodies in the coming steps.
Western blotting usually involves probing the blocked membrane with a primary antibody that targets a specific protein or epitope. After washing the membrane to eliminate any unbound primary antibody, it is treated with a secondary antibody that targets a part of the primary antibody specific to the species. Though enzymatic labels are frequently used in Western blotting, a radioactive label can also be used instead of an enzyme and coupled to the secondary antibody. When the protein of interest is bound by a radioactive antibody, its position on the blot can be identified by exposing the membrane to a sheet of X-ray film, a process known as autoradiography.
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I, Swagatika Sahu (author of this website), have done my master’s in Biotechnology. I have around twelve years of experience in writing and believe that writing is a great way to share knowledge. I hope the articles on the website will help users in enhancing their intellect in Biotechnology.