Western Blot: Overview & Application

Last updated on January 18, 2023 by Excedr

Western Blot: Overview

Western blotting is a technique used in labs to separate and identify specific proteins in tissue, blood samples, or other cell lysates. It’s also known as immunoblotting.

It involves transferring proteins, separated on a gel through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), to the surface of immunoblots (PVDF membranes or nitrocellulose membranes). Before use, the membranes must be soaked in 95% ethanol, methanol, or isopropanol.

The final stage is incubating antibodies specific to the target protein with the protein-containing membrane. Then, the antibody binding activity is identified using detection methods such as chemiluminescent, colorimetric, fluorescent, or radioactive detection. 

Other similar techniques to western blotting are immunocytochemistry, quantitative dot blot, immunohistochemistry, and dot blot analysis. These techniques use antibodies to detect proteins in cells or tissue lysates by using staining methods like enzyme-linked immunosorbent assay (ELISA) or immunostaining. 

The name western blot was given by W. Neal Burnette in 1981 after the already introduced method of Southern blotting, a DNA detection approach. Today, western blotting is a workhorse approach in labs and industries to diagnose diseases, confirm gene expression, demonstrate antibody specificity, and detect post-translational modifications (PTMs).

In this article, we will cover the principle of western blotting, its procedure, and its applications in a range of life sciences lab workflows. 

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How Does Western Blot Work?

Western blot analysis follows the given steps:

  1. Protein from the given sample or lysate is separated electrophoretically using SDS-PAGE. The proteins are separated on the gel based on their molecular weight (usually kilodalton or kDa).

Figure: Protein separation in the form of bands on a Novex Tris-Glycine protein gel, stained with a simple blue safe stain.
  • Using an electric current, the proteins separated on the gel are transferred to a blotting membrane, such as PVDF (polyvinylidene difluoride) or nitrocellulose membrane, forming protein bands. The technique is known as electroblotting.
Figure: A representation of western blot assembly.
  • The transferred proteins on the blotting membrane are stained using the “total protein staining method” for their visualization using imaging systems. It allows the user to verify the uniformity of protein transfer and carry out normalization using loading control, based on the actual amount of protein transferred per lane.
  • The blotting membrane is put into a blocking buffer to prevent the non-specific binding of molecules to the membrane. Then, it’s incubated with a primary antibody specific to the protein of interest followed by a washing step to remove unbound antibodies. 
  • Following incubation with a washing solution, the membrane is incubated again with an enzyme conjugate secondary antibody that binds with the primary antibody. The enzymes mainly used in the process are horseradish peroxidase (HRP) or alkaline phosphatase (AP).
  • Then, incubation of the immunoblot or membrane blot with a substrate specific to the conjugated enzyme leads to the visualization of protein bands of interest from the original mixture of proteins.
Figure: An illustration of the Western Blotting procedure.

To perform a western blot analysis you need high-quality reagents, such as blocking buffers (generally milk or bovine serum albumin [BSA]), wash buffers (such as PBS or TBST, consisting of Tween 20, tris, and NaCl), transfer buffers, primary and secondary antibodies, blotting membranes, and ECL (enhanced chemiluminescence) mix.

What is Western Blot Used For?

The western blotting protocol was developed by Towbin, et al. in 1979 and is now used as a routine procedure in labs for the detection and quantification of proteins. 

Antibody-antigen interactions enable a target protein to be identified in a complex protein mixture due to its specificity. Further, it’s also possible to obtain qualitative and semi-quantitative information about a protein using the Western blotting technique.

Protein Detection

Protein detection through the western blotting technique is a routine workflow in molecular biology and proteomics labs. In this assay, protein bands separated and obtained on a gel through gel electrophoresis are transferred on a blotting membrane using electric current.

Figure: Protein detection using the western blotting method. 

What Industries Use Western Blot?

The western blotting technique is widely used in life sciences, molecular biology, and proteomics labs to detect and quantify proteins, study basic amino acid sequences forming proteins, and quantify protein expression in organisms.

Biotechnology

In biotech labs, it’s a workhorse immunoassay to demonstrate antibody specificity, confirm gene expression, detect post-translational modifications (PTMs), diagnose diseases, study protein-protein interaction, and more. Furthermore, the method is used to identify a single protein in a complex mixture of protein samples.

Pharmaceutical

Western blot analysis has major applications in the medical and pharma industries. For example, 

It detects specific proteins (called HIV antibodies or HIV indicators) present in the blood by separating blood proteins. Further, it’s also used to confirm the results of an ELISA test, which is one of the commonly used immunoassays for the detection of a spectrum of diseases.

Diagnostics

A western blot test is used to detect anti-HIV antibodies in human serum samples. Sample containing proteins from HIV-infected cells are analyzed. The total protein is separated using gel electrophoresis and blotted on the membrane for proper identification of disease indicator proteins.

Furthermore, western blots are used to diagnose variant Creutzfeldt-Jakob Disease. It’s a type of prion disease linked to the consumption of contaminated beef, especially one suffering from bovine spongiform encephalopathy (also called ‘mad cow disease). Western blotting is also a common diagnostic tool in the diagnosis of tularemia.  

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Western blotting is an immunoassay technique used in labs to measure protein concentration, identify specific proteins, and evaluate protein levels in a sample. In the process, proteins are separated in the form of bands using SDS-PAGE followed by electrophoretic transfer of proteins on immunoblots, such as PVDF or nitrocellulose membrane. 

Western blotting has many applications in biotech and medical labs for research and diagnostic purposes. It’s used to study proteins, quantify their concentration in a specific sample, and help in the identification of diseases, such as HIV.

Western blotting is a high-throughput technique, which requires high-quality reagents paired with high-tech equipment to provide reliable and accurate data /output. This makes it challenging for small labs to carry out their research experiments and heavily impacts their budget.

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