Western Blot

Western blotting (WB) has always received extensive attention in biochemical research. After years of accumulation, Creative Biolabs has accumulated rich experience in the optimization and application of WB. We are happy to provide customers with WB technical support for accurate experimental results.

Overview of WB

The study of molecular biology involves many "blotting" techniques, such as southern blot, northern blot, eastern blot, and WB. Every technology is very powerful. For example, southern blots are used to detect DNA sequences, while northern blots are closely related to gene expression. Eastern blotting mainly involves post-translational modification of proteins. Although different techniques are used for different purposes, the principles and processes of these techniques are similar. Among these techniques, WB is the most widely used one. WB is an important technique for identifying and quantifying specific proteins in a given sample, including cell lysates, tissue homogenates, or extracts. With simple protocols and specific reagents, WB enables semi-quantitative visual assessment of targets. WB technology is mainly based on size-based protein separation, followed by the use of antibodies for highly specific detection of the target protein.

WB Definition

WB has wide applications in biochemistry and other sciences because it can detect and characterize a wide variety of proteins. WB is the process of separating proteins and identifying them in complex biological samples. The sensitivity of the process depends on the efficiency of protein transfer retention during processing and final detection. WB works by using SDS-PAGE gel electrophoresis to separate proteins based on their size. Next, the protein is transferred to the membrane by an electric current. After the protein has been transferred, a primary antibody specific for the target protein is added. Next, the secondary antibody with the enzyme is applied, and finally, the substrate for the enzyme reaction bound to the secondary antibody is added for detection and visualization.

Fig.1 The workflow of western blot assay. (Meftahi, 2021)

Advantages of WB

Simple equipment and inexpensive reagents allow WB to be performed with only a small amount of reagents.
Experimental antibodies are readily available as there are thousands of commercially available antibodies.
WB is widely used in many fields because it can detect not only proteins but also other properties of proteins, such as half-life. In addition, WB can detect changes in functional status, such as receptor phosphorylation, methylation.
Sensitivity is the most significant advantage of WB. Because of its ability to detect and characterize small numbers of proteins, the technique could theoretically serve as an effective early diagnostic tool.
WB has higher specificity- the higher the specificity, the more independent the method is from the specificity of the antibody.
The results obtained by WB are also easy to interpret, unique and unambiguous.

Limitations of WB

The tissue used in the experimental procedure must be homogenized.
The technology is an expensive process, the cost of antibodies and detection methods are expensive.
WB is a very delicate process, and any step in the experiment can have an impact on the experimental results.
WB is technically demanding, the process is time-consuming and complex, so it can only be performed by professional personnel.
Small proteins may not be retained by the membrane during the experiment, while larger proteins are difficult to transfer to the membrane.
Subjective judgments are prone to occur. Improper blotting and processing often result in abnormal bands, subjecting test results to subjective interpretation.
Some antibodies may have off-target effects by interacting with more than one protein in the sample.

WB has an irreplaceable position in the field of protein research. Creative Biolabs is committed to providing WB-related optimization solutions and reference information, including but not limited to:

In addition to giving technical guidance to customers on experimentation, we can also provide cost-effective natural autoantibodies (NAA) services to help customers speed up their projects. Please contact us in time for more details.

Reference:

Meftahi, G. H.; et al. Applications of western blot technique: From bench to bedside. Biochemistry and Molecular Biology Education. 2021.

Choosing natural autoantibody (NAA) microarray to profile autoantibody repertoire and reveal novel disease's marker.

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