What Is the Western Blot Technique Used to Detect?

Western Absorb - Lab Technique Used to Find Proteins

Introduction

Western blot is an invaluable lab technique used to detect proteins in a tissue or claret sample. It helps researchers identify specific poly peptide molecules in a complex mixture of proteins. Since antibodies are used in this technique to mark the target protein, this technique is also known as an immunoblot.

Western Blotting

Principle

In Western blot, gel electrophoresis is used to divide proteins in a sample based on their molecular weight. The separated proteins are and so transferred to a solid support, which is and then exposed to antibodies that tin demark to the target protein. This binding is detected using a chemical or radioactive tag. Large protein molecules need to exist denatured before electrophoresis to facilitate their motion in the gel.

Process

The technique involves iv key steps, which are discussed below:

Denaturation of proteins

Denaturing involves unfolding of the protein'due south tertiary structure to a linear structure. Detergents such as sodium dodecyl sulfate are commonly used to denature proteins. Detergents too offer a negative charge to the poly peptide molecules, which boosts electrophoretic mobility.

Separation of proteins

The denatured protein sample is loaded onto an electrophoretic gel and an electrical charge is applied. Protein molecules are separated on the footing of their size and electric charge. Smaller and highly charged molecules movement quickly in the gel and get farther than the bulkier molecules.

Transfer to a support membrane

The separated proteins is transferred to a sheet of blotting paper made of nitrocellulose. The design of the protein molecules in the gel remains the aforementioned in the blotting paper.

Visualizing target protein

A principal or monoclonal antibody is added to the absorb, which binds to the target protein. A labeled secondary antibody which binds to the primary antibody is then added, allowing detection of the specific protein.

Applications of Western Blotting

Some of the important applications of Western blot are listed below:

In the detection of circulating antibodies specific to a unmarried protein or several proteins.
In clinical diagnosis – in HIV testing to detect anti-HIV antibody in the serum sample or as confirmatory tests for diseases such as epidermolysis bullosa acquisita or paraneoplastic pemphigus
In the assay of biomarkers such equally hormones, growth factors, and cytokines
In gene expression studies

Disadvantages

A few limitations of the Western blot technique are listed below:

Western blot is a very frail and fourth dimension-consuming process. A infinitesimal imbalance at whatever level of the procedure can skew the results of the unabridged process.
The secondary anti-trunk can sometimes react with a non-intended protein and this tin can cause labeling of an incorrect protein.
Insufficient transfer time tin can upshot in the larger proteins not transferring properly. This tin can cause erroneous bands or no bands at all.
Well trained technicians are a must for this technique
Western blot is semi-quantitative at best. Simply an approximate estimation and non a precise measurement of molecular weight of the poly peptide is possible
Chief antibiotic availability is crucial. If it is not bachelor for a specific protein, western absorb cannot exist used to detect that protein.

Summary

Western blot is an constructive technique for studying poly peptide expression in the lab. Past stripping the antibody after immunodetection, identification of fifty-fifty multiple poly peptide targets is possible using this technique. However, newer and more than advanced techniques such as immunohistochemistry, menstruum cytometry, and immunofluorescence are more accurate and sensitive compared to the western blot.

These techniques also allow in situ examination of protein expression. The enzyme-linked immunosorbent assay (ELISA) technique is also replacing western blot in many labs as information technology is more rapid and simple compared to the complex western absorb.

One major advantage is that unfolding of the poly peptide's 3D structure is not needed for ELISA, which helps in preserving the immunologic epitopes of proteins. However, ELISA cannot detect multiple target proteins, unlike the western blot.

References

http://world wide web.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
http://world wide web.proteinatlas.org/learn/method/western+blot
http://www.cdc.gov/mmwr/preview/mmwrhtml/00001431.htm
http://www.nature.com/jid/journal/v133/n7/total/jid2013216a.html

Last Updated: May 27, 2019

Written by

Susha Cheriyedath

Susha has a Bachelor of Science (B.Sc.) degree in Chemical science and Master of Science (M.Sc) caste in Biochemistry from the University of Calicut, India. She ever had a cracking involvement in medical and wellness science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare fourth dimension, she loves to cook up a tempest in the kitchen with her super-messy baking experiments.

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