Difference Between Southern Northern and Western Blotting

The main difference between Southern Northern and Western blotting is that the Southern blotting involves the identification of DNA, and the Northern blotting involves the identification of RNA, whereas the Western blotting involves the identification of proteins.

Southern, Northern, and Western are three blotting techniques used to detect a specific DNA, RNA or protein molecule in a sample. During blotting, the macromolecules are transferred onto a membrane from the gel and made to bind with a specific nucleic acid or antibody that aid in the detection.

Key areas Covered

1. What is Southern Blotting
     – Definition, Process, Applications
2. What is Northern Blotting
     – Definition, Process, Applications
3. What is Western Blotting
     – Definition, Process, Applications
4. What are the Similarities Between Southern Northern and Western Blotting
     – Outline of Common Features
5. What is the Difference Between Southern Northern and Western Blotting
     – Comparison of Key Differences

Key Terms

Antibody, DNA, Electrophoresis, Northern Blotting, RNA, Proteins, Southern Blotting, Western Blotting

What is Southern Blotting

Southern blotting is the technique used to detect a specific DNA sequence in a mixture of DNAs. The basic steps of a Southern blot are as below.

Electrophoresis – It separates the DNA sample into distinct bands based on the size by gel electrophoresis.

Transfer – During the transfer, a nitrocellulose membrane is placed in contact with the gel and DNA bands move onto the membrane through the capillary action. 

Detection of Specific Sequences – The target sequence on the membrane is hybridized with the specific, labelled oligonucleotide sequence called the hybridization probe. The hybridization probe is short, 100-500 bp in size, single-stranded DNA molecule. Stringency, which depends on the temperature and the salt concentration of the hybridization buffer, affect hybridization. High stringency defined by the high temperature and low salt concentration increases the specificity of the hybridization while low stringency defined by low temperature and high salt concentration decreases the specificity.

Figure 1: Southern Blotting

The main purpose of Southern blotting is to detect a specific DNA sequence in a sample. The technique is used in DNA fingerprinting, paternity testing, victim identification, and criminal identification. It also helps in the identification of specific genes, in RFLP (restriction fragment length polymorphism), mutation and identification of gene rearrangement, genetic diseases and infectious agents.

What is Northern Blotting

Northern blotting is the technique used to detect a specific RNA in a mixture of RNAs. The basic steps of a Northern Blot are as below.

Electrophoresis – It separates RNA sample according to the size into distinct bands.

Transfer – By capillary action, RNA bands in the gel are transferred onto the membrane.

Detection of specific sequences – The target RNA sequence is detected by hybridization with a labelled oligonucleotide probe made up of DNA.

Figure 2: Northern Blotting

Since Northern blotting can identify specific RNA sequence in a sample, it can be used in the gene expression studies. It can also help in disease diagnosis.

What is Western Blotting

Western blot is a blotting technique used to detect a specific amino acid sequence in a mixture of proteins. The basic steps of a Western Blot are as below.

Electrophoresis – Individual proteins are separated based on the size into bands by SDS PAGE.

Transfer – Protein bands in the gel are transferred onto a membrane by blotting.

Detection of Specific Proteins – The membrane with separated proteins is incubated with a primary antibody that only bind to the specific protein. A secondary antibody, which is labeled with an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase, is used to detect the primary antibody. When incubated with the substrate, the enzyme action visualizes the binding of antibodies to a specific region on the membrane.

Figure 3: Western Blotting

Western blotting is also called protein blot or immunoblotting. It can identify the number of proteins in a sample, presence of bacteria and virus in serum, and the presence of HIV antibodies in the serum. It can also detect defective proteins. Moreover, western blotting is the definitive test for Hepatitis B, Creutzfeldt-Jacob disease, Lyme disease, and Herpes.

Similarities Between Southern Northern and Western Blotting

• Southern blotting, Northern blotting, and Western blotting are three blotting techniques used to identify a particular type of macromolecule within a sample.
• The three steps of the blotting techniques are electrophoresis, transfer, and detection.
• Each technique requires denaturation and blocking with the excess, corresponding macromolecule.
• Each technique has applications in biotechnology and medicine.

Difference Between Southern Northern and Western Blotting

Definition

Southern blotting refers to a procedure for identifying specific sequences of DNA, while Northern blotting refers to an adaptation of the Southern blot procedure used to detect specific sequences of RNA by hybridization with complementary DNA and Western blotting refers to a blotting procedure used to identify specific amino-acid sequences in proteins.

Developed by

Southern blotting was developed by Edward M. Southern in 1975. Northern blotting was developed by Alwine and his colleagues in 1979, and Western blotting was developed by George Stark’s group at Stanford University in 1979.

Molecule Detected

Southern blotting detects specific DNA sequences, Northern blotting detects specific RNA sequences, while Western blotting detects specific proteins.

Gel Electrophoresis

Southern blotting involves Agarose gel electrophoresis, and Northern blotting involves denaturing formaldehyde agarose gel while Western blotting involves SDS PAGE.

Blotting Method

Both Southern and Northern Blotting involve capillary transfer while Western blotting involves an electric transfer.

Probes

Southern blotting uses DNA probes while Northern blotting uses cDNA probes and Western blotting uses primary and secondary antibodies.

Application

Southern blotting is used to identify specific gene sequences and in DNA fingerprinting. Northern blotting is used in gene expression analysis, and Western blotting is used in disease diagnosis.

Conclusion

Southern blotting identifies specific DNA sequence, while Northern blotting identifies specific RNA sequence and Western blotting identifies specific protein sequence in a sample. Therefore, the main difference between the three blotting techniques is the type of macromolecule they detect.

Reference:

1. “Southern Blotting: Principle, Procedure and Application -.” ONLINE BIOLOGY NOTES, 4 Dec. 2017, Available Here
2. Kochunni, Deena T, and Jazir Haneef. “Northern Blotting: Principle, Procedure and Applications.” Biology Exams 4 U , Available Here
3. “Western Blotting Technique: Principle, Procedure and Application -.” BIOLOGY ONLINE NOTES, 4 Dec. 2017, Available Here

Image Courtesy:

1. “Figure 17 01 05” By CNX OpenStax –  (CC BY 4.0) via Commons Wikimedia
2. “Northern blot diagram” By Ilewieszoośmiornicach – Own work (CC BY-SA 4.0) via Commons Wikimedia   
3. “Western Blotting” By Cawang – Own work (CC0) via Commons Wikimedia