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Western blotting vs Southern blotting, principle and methodology

Overview

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Source: Microbioz India

Date: 31 Jul,2023

Among the key techniques used in molecular biology are the separation of proteins and nucleic acids. There are several methods that may be used, such as western blotting and southern blotting, to identify these macromolecules. The explanation of several biological processes has been made possible by these approaches during the past several years. Although the fundamental mechanisms for each of these systems included the separation of macromolecules (DNA, RNA, and protein) based on their molecular weights. However, they each suggested a unique mechanism for separation, using various biomolecules. In this article, Microbioz aims to dive into the specific differences amongst Western and Southern blotting, as well as to shed light on their technique and uses.

Principle and methodological based difference between Western and Southern blotting

Western blotting:

Western blotting is also referred to as “protein immunoblotting,” which resolves individual proteins present in the sample. A solid support membrane, such as nitrocellulose, nylon, or polyvinylidene fluoride (PVDF), is used to immobilise the replicate molecules after the protein has been separated using PEG (polyacrylamide gel electrophoresis). Additionally, a probe for targeted immobilised molecules is required for antibody-based detection, where secondary antibody and primary antibody binding facilitates the formation of a particular signal. Membrane treatment with a blocking agent (proteins and detergent) is required to prevent nonspecific binding. In addition, the nature of the probe is taken into account while detecting it. For example, radioactive probes are detected using X-ray and phosphorimaging devices, and colorimetric, chemiluminescent, and fluorescence imaging documentation is used for chemical and enzyme-based detection.

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Southern blotting:

The process of Southern blotting, also known as nucleic acid hybridization, involves the use of restriction enzyme to cleave DNA fragments of a defined size. This technique was developed to resolve DNA fragments of a defined size in the form of discrete bands among a complex mixture of other DNA molecules through agarose gel electrophoresis. Next, the replica of the molecules is transferred to a solid support membrane, where it is then hybridised with the DNA probe. In genetic research, gene mapping, DNA fingerprinting, and the detection of genetic mutations or changes in gene copy number, the southern blotting technique is primarily employed.

Various aspects representing difference between western and southern blotting

Aspect	Western blotting	Southern blotting
Target biomolecules	Protein analysis and detection	DNA sequence analysis and detection
Probe used	Antibody probe	DNA probe
Electrophoresis	Polyacrylamide gel electrophoresis	Agarose gel electrophoresis
Transfer method	Electroblotting	Capillary transfer
Target detection	Protein bands	DNA bands
Detection method	Chemiluminescent imaging, fluorescent imaging, colorimetric imaging and phosphorimaging	Radioactive signals or autoradiography
Sensitivity	Highly sensitive	Moderately sensitive
Time requirement	Faster process	Time taking process
Applications	Disease diagnosis and drug development	DNA fingerprinting, genetic mutation identification and gene mapping

Conclusion:

Based on their specificity and sensitivity, Western blotting and southern blotting are the two main molecular biology methods that have had a substantial influence on life science research. These methods are still exceptionally useful for figuring out the molecular causes of various illnesses, looking into genetic diversity, and coming up with novel therapeutic strategies. These fundamental methods continue to be necessary for future developments in biotechnology and other fields as technology develops.