Northern Blotting technique is used for 8th May 2022 – Tags: , ,

Northern blotting technique is a technique used to study gene expression. It is performed by the detection of a specific RNA (or isolated mRNA). The mRNA is usually represented in 5% of the total RNA sequence. This method reveals the identity, number, activity and size of the particular gene.

Northern Blotting Technique

This blotting technique can also be used for the growth of a tissue or organism. At different stages of differentiation and morphogenesis, the abundance of an RNA changes, which can be identified by this technique. It can also identify abnormal, diseased or infected states at the molecular level. The northern blot technique was developed in 1977 by James Alwine, David Kemp and George Stank at Stanford University. The technique was named after the similarity of the process to the southern blot. The main difference between the two techniques is that in the north only RNA is involved in the staining.

The subtypes of blotting, such as northern, western and southern, depend on the target molecule being searched. When a DNA sequence is the base or code for a protein molecule, the particular DNA molecule of interest can be marked by Southern Blotting. In gene expression, when DNA is expressed as mRNA for the production of a protein, this process can be identified by Northern blotting. Finally, when the encoded mRNA produces the protein of interest, this protein identification can be done by Western blotting.

General drying procedure

  • Homogenize the sample.
  • Separation of the molecule of interest using an electrophoresis membrane.
  • Transfer of the molecules to a nitrocellulose/nylon membrane.
  • Hybridization or identification of the molecule.

Northern Blotting Technique is used for

Northern blotting technique is an analytical technique used in biology and chemistry to detect specific RNA from a mixture of RNAs. The technique was developed by James Alwine in 1971 at Stanford University.

In this technique :

  • RNAs are denatured by breaking H-bonds through the application of formaldehyde (not through the application of a restriction endonuclease, as is done in Southern blotting).
  • RNAs are then immobilised on a nylon or nitrocellulose membrane after separation by electrophoresis.
  • Detection is done by radioactively labelled probes, e.g. P-32, under an autoradiogram or non-radioactive probes (bioluminescent or enzyme-linked).
northern blotting technique 2022
Northern blotting steps

Northern blotting steps

The Southern blotting technique consists of four basic steps:

In the first step, the DNA sample is split or digested into small pieces using a restriction enzyme. After digestion, the DNA fragments are separated by gel electrophoresis. An agarose gel is normally used for this purpose. The electrophoresis shows several bands that look like a blot due to the presence of several small restriction fragments in the gel. NaOH is then used to denature the DNA into single strands.

These bands are then transferred to the surface of a membrane using an electrical gradient which is usually a sheet of paper called blotting paper. The pattern of DNA fragments on the gel remains the same after transfer to the blotting paper.

A probe consisting of single-stranded DNA fragments is then introduced into the blotting paper. The bases of the DNA probe will be paired with complementary DNA sequences in the gel to form the double-stranded DNA. The probe is usually labelled with a chemical or radioactive label to allow tracking of the probe in the gel. Chemical substrates and X-ray films are used to locate the probe if the label is an enzyme. Radioactive labels can appear directly on X-ray films.

Principle

Like all normal blotting techniques, Northern blotting begins with electrophoresis to separate RNA samples according to size. Electrophoresis separates RNA molecules according to the charge of the nucleic acids. The charge of the nucleic acids is proportional to the size of the nucleic acid sequence.

Thus, the electrophoresis membrane separates the nucleic acid sequence according to the size of the RNA sequence. In cases where our target sequence is an mRNA, the sample can be isolated by oligocellulose chromatography techniques, since mRNAs are characterised by the poly(A) tail. As the gel molecules are fragile in nature, the separated sequences are transferred to nylon membranes. The choice of nylon membrane is due to the fact that nucleic acids are negatively charged by nature. Once the RNA molecules are transferred, they are immobilised by a covalent bond. The probe is then added, the probe may be complementary to an ssDNA sequence. Formamide is generally used as a blotting buffer as it reduces the annealing temperature.

northern blotting applications

Techniques such as real-time PCR allow very reliable and rapid detection of even a small sequence of targets, whereas Southern blotting requires a large amount of target DNA. In addition, newer techniques such as fluorescence in situ hybridization (FISH) allow very sensitive identification of specific nucleotide sequences in a tissue sample with precise localization. Real-time PCR and FISH also allow accurate quantification of the target, which cannot be fully achieved by Southern blotting.

Southern blot has several applications in today’s molecular biology laboratory. Some of the main applications of Southern blot are

  • Identification of a single gene in a set of DNA fragments
  • Genetic mapping
  • Analysis of DNA gene patterns
  • Detection of specific DNA sequences in a genome
  • the study of gene deletions, duplications and mutations that cause various diseases
  • detecting genetic diseases and cancers, such as monoclonal leukemia and sickle cell mutations
  • detecting the presence of a gene family in a genome
  • genetic fingerprinting and forensic testing, such as paternity testing and sex determination.

Western Blot

Western blot technique determines the molecular weight of proteins and measures the abundance of proteins in different samples.

Features Western Blot :

  • After separation by gel electrophoresis using the SDS-PAGE method, the proteins are transferred to a special blotting paper called nitrocellulose, although other types of paper, or membranes, can be used. The proteins retain the same separation pattern as they had on the gel.
  • The blot is incubated with a generic protein (such as a milk protein) to bind to any sticky spots remaining on the nitrocellulose. An antibody capable of binding to its specific protein is then added to the solution. The antibody is conjugated to alkaline phosphatase or horseradish peroxidase.
  • The position of the antibody is revealed by incubating a colourless substrate which the bound enzyme converts into a coloured product that can be seen and photographed.

Western blot (sometimes called protein immunoblot) is a widely recognised analytical technique used to detect specific proteins in a given homogenised sample or tissue extract. Western blot samples can be taken from whole tissues or from cell cultures. Solid tissues are first mechanically ground using a blender (for large sample volumes), a homogeniser (for small volumes) or by sonication. Detergents, salts and various buffers can be used to stimulate cell lysis and solubilise the proteins.

This technique uses gel electrophoresis to separate native proteins according to their three-dimensional structure or denatured proteins according to polypeptide length.

Western Blot

Western Blot protocol

Western blotting (WB) is a commonly used technique in molecular and cell biology as well as protein engineering. Using a WB assay, researchers can separate and identify specific proteins from a complex mixture of protein extractions in cells. There are three main parts to this task: separation by molecular size, transfer to a solid support by gel electrophoresis, and incubation of the target protein with appropriate primary and secondary antibody labels for visualisation.

WESTERN BLOTTING WORKFLOW STEPS:

  1. Sample preparation: cell lysis and protein extraction
    SDS-PAGE gel electrophoresis
  2. Membrane transfer
    a) Transfer from the tank
    b) Semi-dry transfer
  3. Immunodetection
    a) Traditional immunoassay protection
    b) 30-minute immunoassay protocol with SNAP i.d.
    c) Immunodetection of gait with Immobilon GO
Western Blot protocol

Many antibody assay techniques, such as indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), have been developed and revised in the past. Although sensitive, these methods are also subject to cross reactions that make weak positive results difficult to interpret.

Difference Between Southern Northern & Western Blotting

The main difference between south-north and west staining is that south staining involves the identification of DNA, and north staining involves the identification of RNA, while west staining involves the identification of protein.

South, north and west blotting are three blotting techniques used to detect a specific DNA, RNA or protein molecule in a sample. During blotting, macromolecules are transferred from the gel to a membrane and bind to a specific nucleic acid or antibody that facilitates detection.