Difference between Northern Southern and Western Blotting

Different blots are used to identify the existence of one particular target molecule, such as DNA, RNA or
protein, in an intricate mixture of associated molecules. Blotting is the process of transferring
macromolecules (proteins, nucleic acids, etc.) from a gel to the solid surface of an immobilised
membrane to detect the molecules that have been transferred.

Identification of specific sequences of DNA, RNA, and proteins is important for various studies in
Molecular biology. Gel electrophoresis is a process that segregates DNA, RNA, and proteins based on
their molecular weights. Specific nucleic acid or protein sequences are identified from the gel profiles
using specialised blotting and hybridisation procedures with tagged probes.

The key distinction between Northern, Southern and Western blotting depends on the type of molecule
it identifies from a sample. The Southern blotting method and the Northern blotting method can be used
to identify specific DNA and RNA sequences, respectively, while the Western blotting method can be
used to identify a specific protein.

Table of Contents

Northern Blotting

Southern Blotting

Western Blotting

Difference between Northern Southern and Western Blotting

Frequently Asked Questions (FAQs)

Northern Blotting

Northern blotting is the method used to detect a specific RNA sequence in a sample of mixed RNAs.
Below are the basic steps in a Northern Blot.

Electrophoresis – It segregates RNA samples based on their size into different bands.

Transfer – RNA bands in the gel are moved to the membrane by capillary action.
Identification of specific sequences – The target RNA sequence is identified by hybridisation with a
tagged oligonucleotide probe made of DNA.

Northern blotting can be utilised in gene expression research because it can identify a specific RNA
sequence in a sample. It can also assist in the diagnosis of diseases.

Southern Blotting

Southern blotting is the method for detecting a specific DNA sequence in a sample of mixed DNA. Below
are the basic steps in a Southern blot.

Electrophoresis – It segregates the DNA sample into separate bands based on their size by gel
electrophoresis.

Transfer – DNA bands are transferred by capillary action onto a nitrocellulose membrane in contact with
the gel during the transfer process.

Identification of specific sequences – The particular, tagged oligonucleotide sequence known as the
hybridisation probe (100–500 bp) is hybridised with the membrane’s target sequence. Stringency has an
impact on hybridisation and is influenced by the temperature and salt content of the hybridisation
buffer. Low stringency is defined as low temperature and high salt concentration, reducing the
specificity of hybridisation, while high stringency is defined as high temperature and low salt
concentration, which increases the specificity.

Western Blotting

A particular amino acid sequence can be identified in a mixture of proteins using the Western blotting
technique. Below are the basic steps of a Western Blot.

Electrophoresis – Individual proteins can be separated based on their size into separate bands using SDS
PAGE.
Transfer – By blotting, protein bands in the gel are moved from the membrane to the gel.

Identifying particular proteins – A primary antibody binding to a particular protein is incubated on a

membrane with isolated specific proteins. The primary antibody is identified using a secondary antibody
that has been tagged with an enzyme, such as alkaline phosphatase or horseradish peroxidase (HRP).
The enzyme action, incubated with the substrate, shows how antibodies bind to a specific area on the
membrane.

Difference between Northern, Southern and Western Blotting

Northern Blotting

Southern Blotting

Western Blotting

Northern blot is used to detect a specific RNA sequence in a sample of mixed RNAs

Southern blot is used for detecting a specific DNA sequence in a sample of mixed DNA

Western blot is used to identify a specific amino acid sequence in a sample of mixed proteins

A specific RNA sequence is detected

A specific DNA sequence is detected

A specific amino acid sequence is detected

This is a capillary transfer blotting technique

This is also a capillary transfer blotting technique

This is an electric transfer blotting technique

cDNA or RNA probes are used with radioactive or non-radioactive labels

Single-stranded DNA or RNA is used as a probe

Antibodies against antigen epitopes are used as the probe

Detection mechanism include chemiluminescence, X-Ray film

Detection mechanism include chemiluminescence, X-Ray film

Detection mechanism include CCD camera, Film, LED or infrared imaging mechanism

It was developed by Alwine and his colleagues in 1979

It was developed by Edward M. Southern in 1975

It was developed by George Stark’s group at Stanford University in 1979

Northern blotting encompasses denaturing formaldehyde agarose gel

Southern blotting encompasses Agarose gel electrophoresis

Western blotting encompasses SDS PAGE

It can be used in gene expression studies and can assist in disease diagnosis

The method is used in the identification of criminals, victim identification, paternity testing, and DNA
fingerprinting

It can determine the quantity of proteins in a sample, the presence of bacteria and viruses in the serum,
and the presence of antibodies to HIV. It can also identify defective proteins

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What is meant by Eastern blotting, Western blotting, Southern blotting and Northern blotting?

Frequently Asked Questions – FAQs

Which enzyme is most often used in blotting procedures?

Horse-reddish peroxidase or phosphatase-labelled secondary antibodies are the most widely used
enzyme-labelled secondary antibodies because they produce colourful products when employed with
the substrate.

Which gel is used in Northern blotting?

Northern blotting encompasses denaturing formaldehyde agarose gel.

What is the main purpose of blotting?

Blotting is a common diagnostic technique used in molecular biology to identify proteins and nucleic
acids. This method immobilises the target molecule on a support, such as a nylon or nitrocellulose
membrane