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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Related Links:
- What is meant by Eastern blotting, Western blotting, Southern blotting and Northern blotting?
Frequently Asked Questions – FAQs
Q1
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.
Q2
Which gel is used in Northern blotting?
Northern blotting encompasses denaturing formaldehyde agarose gel.
Q3
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.
