Key Difference – Northern vs Southern vs Western Blotting
Detection of specific sequences of DNA, RNA, and proteins is essential for various types of studies in Molecular biology. Gel electrophoresis is a technique which separates DNA, RNA, and proteins according to their sizes. From the gel profiles, particular DNA sequence, RNA sequence, or protein are detected by the special techniques called blotting and hybridization with labeled probes. There are three different types blotting methods namely southern, northern and western. The key difference between northern southern and western blotting lies with the type of the molecule it detects from a sample. Southern blotting is a method which detects a specific DNA sequence from a DNA sample. Northern blotting is a technique which detects a specific RNA sequence from a RNA sample. Western blotting is a method which detects a specific protein from a protein sample.
1. Overview and Key Difference
2. What is Southern Blotting
3. What is Northern Blotting
4. What is Western Blotting
5. Side by Side Comparison – Northern vs Southern vs Western Blotting
What is Southern Blotting?
Southern blotting technique was developed by E. M. Southern in 1975 for the identification of a specific DNA sequence from a DNA sample. This is the first blotting technique introduced in molecular biology. It enabled the detection of specific genes from the DNA, specific fragments from DNA, etc. There are several steps involved in southern blotting technique. They are as follows.
- DNA is isolated from the sample and digested with restriction endonucleases.
- Digested sample is separated by Agarose gel electrophoresis.
- DNA fragments in the gel are denatured into single strands by the use of an alkaline solution.
- Single stranded DNA is transferred to a nitrocellulose filter membrane by capillary transferring.
- Transferred DNA is fixed onto the membrane permanently.
- Fixed DNA on the membrane is hybridized with labeled probes.
- Unbound DNA is washed off from the membrane by washing.
- X-ray film is exposed to the membrane and an autoradiograph is prepared.
Southern blotting is applied for different aspects of molecular biology. It is useful in RFLP mapping, forensic studies, DNA methylation in gene expression, detection of mutated genes in genetic disorders, DNA fingerprinting, etc.
What is Northern Blotting?
Northern blotting is a method designed to detect a specific RNA sequence or mRNA sequence from a sample to study gene expression. This technique was developed by Alwine, Kemp, and Stark in 1979. It differs from the sourthern and western blotting techniques due to several steps. However, this technique is also performed via gel electrophoresis, blotting, and hybridization with specific labeled probes and detection. Northern blotting technique is performed as follows.
- RNA is extracted from the sample and separated by gel electrophoresis.
- RNA is transferred from the gel onto a blotting membrane and fixed.
- The membrane is treated with a labeled probe prepared from cDNA or RNA ( the probe is complementary to a specific sequence in the sample).
- The probe is incubated with the membrane to bind with a specific sequence.
- Unbound probes are washed off.
- Hybridized fragments are detected by an autoradiograph.
Northern blotting is an important tool in detection and quantification of hybridized mRNA, studying RNA degradation, evaluation of RNA half-life, detection of RNA splicing, studying gene expression, etc.
What is Western Blotting?
Western blotting is a method of detecting a specific protein from a protein mixture by the use of labeled antibody. Therefore, western blot is also known as an immunoblot. This technique was introduced by Towbin et al in 1979 and it is now routinely performed in the labs for protein analysis. Steps are as follows.
- Proteins are extracted from the sample
- Proteins are separated by their sizes using polyacrylamide gel electrophoresis
- Separated molecules are transferred into a PVDF membrane or nitrocellulose membrane by electroporation
- The membrane is blocked for nonspecific binding with the antibodies
- Transferred proteins are bound with primary antibody (enzyme labeled antibodies).
- The membrane is washed to remove nonspecifically bound primary antibodies
- Bound antibodies are detected by adding a substrate and detecting the coloured precipitate formed
Western blotting is useful in the detection of anti-HIV antibodies in a human serum sample. Western blot can also be used as a confirmatory test for Hepatitis B infection and definitive test for mad cow disease.
What is the difference between Northern Southern and Western Blotting?
Northern vs Southern vs Western Blotting
|Type of Molecule Detected|
|Northern Blotting||Northern blotting detects a specific RNA sequence from an RNA sample.|
|Southern Blotting||Southern blotting detects a specific DNA sequence from a DNA sample.|
|Western Blotting||Western blotting detects a specific protein from a protein sample.|
|Type of Gel|
|Northern Blotting||This uses Agarose/formaldehyde gel.|
|Southern Blotting||This uses an Agarose gel.|
|Western Blotting||This uses Polyacrylamide gel.|
|Northern Blotting||This is a capillary transfer.|
|Southern Blotting||This is a capillary transfer.|
|Western Blotting||This is an electric transfer.|
|Northern Blotting||cDNA or RNA probes labeled radioactively or nonradioactively.|
|Southern Blotting||DNA probes are labeled radioactively or non-radioactively.|
|Western Blotting||Primary antibodies are used as probes.|
|Northern Blotting||This is done using an autoradiograph, or detection of light or color change.|
|Southern Blotting||This is done using an autoradiograph, detection of light or color change.|
|Western Blotting||This is done using the detection of light or color change.|
Summary – Northern vs Southern vs Western Blotting
Blotting is a special technique developed for the identification of specific DNA, RNA or protein from the samples. There are three separate blotting procedures, namely northern, southern and western, to detect a specific type of molecule. Northern blotting technique is designed to detect a specific RNA sequence from a mixture of RNA. Southern blotting technique enables the detection of a specific DNA sequence from a DNA sample and western blotting technique is developed to identify a specific protein from a protein mixture.
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2. Brown, T. “Southern blotting.” Current protocols in immunology. U.S. National Library of Medicine, May 2001. Web. 27 Mar. 2017
3. He, Shan L. “Northern blot.” Methods in enzymology. U.S. National Library of Medicine, 2013. Web. 27 Mar. 2017