Key Difference – Northern vs Southern vs Western Blotting
Detection of specific sequences of DNA, RNA, and proteins is crucial for assorted types of studies in Molecular biology. Gel electrophoresis is a method which sepaprices DNA, RNA, and also proteins according to their sizes. From the gel prorecords, certain DNA sequence, RNA sequence, or protein are detected by the one-of-a-kind methods referred to as blotting and also hybridization with labeled probes. There are 3 different forms blotting approaches namely southern, northern and western. The essential difference between northern southerly and also western blotting lies with the type of the molecule it detects from a sample. Southern blotting is a technique which detects a details DNA sequence from a DNA sample. Northern blotting is a technique which detects a particular RNA sequence from a RNA sample. Western blotting is a method which detects a certain protein from a protein sample.
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CONTENTS1. Synopsis and also Key Difference2. What is Southern Blotting3. What is Northern Blotting4. What is Western Blotting5. Side by Side Comparison – Northern vs Southern vs Western Blotting6. Summary
What is Southern Blotting?
Southern blotting technique was developed by E. M. Southern in 1975 for the identification of a particular DNA sequence from a DNA sample. This is the initially blotting technique introduced in molecular biology. It permitted the detection of particular genes from the DNA, certain pieces from DNA, and so on. Tbelow are several steps associated in southerly blotting method. They are as complies with.DNA fragments in the gel are denatured into single strands by the usage of an alkaline solution.Single stranded DNA is moved to a nitrocellulose filter membrane by capillary moving.Transferred DNA is solved onto the membrane permanently.Fixed DNA on the membrane is hybridized via labeled probes.Unbound DNA is waburned off from the membrane by washing.X-ray film is exposed to the membrane and also an autoradiograph is all set.
Southern blotting is applied for various elements of molecular biology. It is useful in RFLP mapping, forensic researches, DNA methylation in gene expression, detection of mutated genes in hereditary disorders, DNA fingerprinting, and so on.
Figure 01: Southern Blotting Technique
What is Northern Blotting?
Northern blotting is an approach designed to detect a specific RNA sequence or mRNA sequence from a sample to study gene expression. This method was arisen by Alwine, Kemp, and Stark in 1979. It differs from the sourthern and western blotting approaches due to a number of steps. However, this approach is additionally performed via gel electrophoresis, blotting, and hybridization with particular labeled probes and detection. Northern blotting method is performed as adheres to.RNA is extracted from the sample and also separated by gel electrophoresis.RNA is moved from the gel onto a blotting membrane and also solved.The probe is incubated via the membrane to bind via a specific sequence.Unbound probes are wamelted off.Hybridized fragments are detected by an autoradiograph.
Northern blotting is an essential tool in detection and quantification of hybridized mRNA, examining RNA deterioration, evaluation of RNA half-life, detection of RNA splicing, studying gene expression, etc.
Figure 02: Northern Blotting
What is Western Blotting?
Western blotting is an approach of detecting a details protein from a protein mixture by the usage of labeled antibody. Thus, western blot is also known as an immunoblot. This approach was presented by Towbin et al in 1979 and it is currently consistently perdeveloped in the labs for protein evaluation. Steps are as complies with.Proteins are extracted from the sampleProteins are separated by their sizes utilizing polyacrylamide gel electrophoresisSeparated molecules are transferred into a PVDF membrane or nitrocellushed membrane by electroporationThe membrane is blocked for nonparticular binding through the antibodiesTransferred proteins are bound with main antibody (enzyme labeled antibodies).The membrane is wamelted to rerelocate nonparticularly bound major antibodiesBound antibodies are detected by including a substprice and detecting the coloured precipitate formed
Western blotting is helpful in the detection of anti-HIV antibodies in a huguy serum sample. Western blot have the right to additionally be supplied as a confirmatory test for Hepatitis B infection and definitive test for mad cow illness.
Figure 03: Western Blotting
Northern vs Southern vs Western Blotting
|Type of Molecule Detected|
|Northern Blotting||Northern blotting detects a certain 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 details protein from a protein sample.|
|Type of Gel|
|Northern Blotting||This offers Agarose/formaldehyde gel.|
|Southern Blotting||This supplies an Agarose gel.|
|Western Blotting||This uses Polyacrylamide gel.|
|Northern Blotting||This is a capillary move.|
|Southern Blotting||This is a capillary deliver.|
|Western Blotting||This is an electric transfer.|
|Northern Blotting||cDNA or RNA probes labeled radioproactively or nonradioproactively.|
|Southern Blotting||DNA probes are labeled radioproactively or non-radioproactively.|
|Western Blotting||Primary antibodies are offered as probes.|
|Northern Blotting||This is done utilizing an autoradiograph, or detection of light or shade change.|
|Southern Blotting||This is done utilizing an autoradiograph, detection of light or color readjust.|
|Western Blotting||This is done utilizing the detection of light or shade readjust.|
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Outline – Northern vs Southern vs Western Blotting
Blotting is a distinct technique arisen for the identification of specific DNA, RNA or protein from the samples. There are three separate blotting procedures, namely northern, southerly and western, to detect a certain kind of molecule. Northern blotting method is designed to detect a specific RNA sequence from a mixture of RNA. Southern blotting strategy permits the detection of a certain DNA sequence from a DNA sample and western blotting approach is occurred to recognize a particular protein from a protein mixture.
References1. Gibbons, Janay. “Western Blot: Protein Transfer Rundown.” North American Journal of Medical Sciences. Medknow Publications & Media Pvt Ltd, Mar. 2014. Internet. 27 Mar. 20172. Brvery own, T. “Southern blotting.” Current protocols in immunology. U.S. National Library of Medicine, May 2001. Internet. 27 Mar. 20173. He, Shan L. “Northern blot.” Methods in enzymology. UNITED STATE National Library of Medicine, 2013. Web. 27 Mar. 2017
Image Courtesy:1. “Northern Blot Scheme” By RNA405 – Own occupational (Public Domain) by means of Commons Wikimedia2. “Western blot 114A” By Amanthabagdon – Own job-related (Public Domain) by means of Commons Wikimedia
Filed Under: Molecular Biology Tagged With: Compare Southern Northern and Western Blotting, immunoblot, Northern Blotting, Northern Blotting Definition, Northern Blotting Steps, Southern Blotting, Southern Blotting Definition, Southern Blotting Steps, Southern Northern and also Western Blotting Differences, Southern Northern vs Western Blotting, Western blotting, Western Blotting Definition, Western Blotting Steps
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