Blotting Technique – Definition, Types, Application

What is Blotting technique?

In molecular biology and genetics, blotting is an analysis technique used for the detection of specific biomolecules (proteins, DNA or RNA) in samples of complex composition by transferring them onto a carrier such as a nitrocellulose, polyvinylidene fluoride or nylon membrane.

• In most cases, the Blotting technique is performed after gel electrophoresis by transferring the molecules from the gel to the blotting membrane or often they are transferred directly onto the membrane.
• After transferring, the proteins, DNA or RNA molecules are visualized by using colorant staining such as silver staining of proteins or by autoradiographic visualization of radiolabelled molecules (performed before the blot), or specific labeling of some proteins or nucleic acids.
• After that, antibodies or hybridization probes are binds only to specific molecules of the blot and have an enzyme joined to them.
• After washing the blot, it incubation with a proper reactive as result it will visualize the enzymatic activity (and so, the molecules we search in the blot). Performing either a colored film on the blot or a chemiluminescent reaction which is recorded by photographic film.
• This method is an adjunct to gel electrophoresis which involves the separation of DNA, RNA and proteins with exceptional resolving power.
• The blotting technique mainly applied to those biomolecules which are stably adhered to nitrocellulose, nylon or paper membrane and are still capable to bind their cognate ligand.
• Before performing the transfer of biomolecules, they are must be separated according to their size range.
• There are different types of blotting techniques, among them, three are important such as, Southern blotting, western blotting, and Northern blotting.
• The blotting method was first discovered by Edwin M. Southern in 1975 for DNA. In his method, the DNA restriction fragments which are electrophoretically fractionated in an agarose gel are transferred to a solid support (nitrocellulose) and recognized as discrete bands following hybridization to a complementary DNA probe.
• When the Southern blotting method was applied to RNA, it was known as Northern Blotting. 
• In Western blotting, the proteins are transferred to a membrane and their detection was done by antibody probe.
• All these blotting methods follow the common steps, which involve the transfers of molecules from the gel to a porous membrane, most often performed by soaking solution through the gel and the membrane applying absorptive paper.
• In blotting the detection of a specific sequence on DNA and RNA in the membrane is done by performing molecular hybridization with labeled nucleic acid probes.
• The proteins are mainly detected by using labeled antibodies.
• The original protocol was adapted for radioactive probes labeled with, for example, 32P, 3H,35S or 125I. Since then, other labeling systems have been developed, including fluorescent and chemiluminescent reagents.

Blotting Overview

Blotting is a widely employed molecular biology technique. There are present different types of blotting such as southern, western, northern and eastern. These types of blottings are used for different macromolecules like lipids, RNA, DNA and proteins. 

All of these blotting techniques are dependent on two factors such as the size of the molecule and their binding ability to the solid support. And lastly, the labeled probes are used to detect the molecule of interest.

During botting first the gel electrophoresis of RNA and DNA or proteins are performed. After that they are transferred onto a specific membrane such as nitrocellulose PVDF or nylon membrane. Sometimes they are directly transferred onto the membrane.

Next, these transferred molecules are visualized by using different staining such as Ethidium bromide, Crystal violet, Safranine and Ossmium tetroxide etc.

Blotting Types

There are mainly four types of blotting such as;

1. Southern blotting
2. Western blotting
3. Northern blotting
4. Eastern blotting

1. Southern blotting

This method was named after Edward M. Southern and only used for the analysis of DNA sequences. The southern blotting is completed in these following steps;

1. First of all the Large DNA is fragmented into small pieces by usign Restriction endonucleases.
2. This fragmented DNA are separated according to their size by using gel electrophoresis.
3. The Larger DNA fragments are treated with HCl, which causes depurination of DNA fragments.
4. After electrophoresis, the separated fragments are transferred onto a nitrocellulose sheet. The pressure is applied over the membrane to confirm the proper interaction occur between these two gel and membrane.
5. The membrane is exposed to ultraviolet radiation as result the fragments weill permanently attached to the membrane.
6. Then the membrane is exposed to a hybridization probe. But the DNA probe is labeled so that it can easily detect when the molecule is tagged with a chromogenic dye.
7. The membrane is washed using an SSC buffer to remove the excess probe.
8. Finally the membrane is visualized on X-ray film with the help of autoradiography.

Southern blotting Applications

Southern blotting has several applications in molecular biology, including:

1. Gene mapping: Southern blotting can be used to map the location of specific genes within a genome. By comparing the hybridization patterns of DNA from different individuals or species, researchers can identify similarities and differences in gene organization and function.
2. DNA fingerprinting: Southern blotting can be used to generate DNA fingerprints, which are unique patterns of DNA fragments that can be used to identify individuals or organisms. This technique has important applications in forensic science and paternity testing.
3. Detecting gene mutations: Southern blotting can be used to detect mutations in specific genes that may be associated with disease or genetic disorders. By comparing the hybridization patterns of DNA from affected and unaffected individuals, researchers can identify the presence of mutations and their potential role in disease.
4. Studying gene expression: Southern blotting can be used to study gene expression by detecting the presence and abundance of specific mRNA transcripts in a sample. This technique, known as Northern blotting, is similar to Southern blotting but uses RNA instead of DNA.

Overall, Southern blotting is a valuable tool in molecular biology that has a wide range of applications in research and diagnostics.

2. Western blotting

This blotting technique was named after W. Neal Burnette and used for the detection and analysis of protein in a given sample. The  Western blotting is completed in the following steps;

1. First of all, the desired proteins are isolated from the particular sample.
2. The beta- mercaptoethanol (BME) and Sodium dodecyl Sulfate (SDS) is added to the protein suspension.
3. After that, the protein- SDS complex is loaded on the top of the gel in the well. A molecular weight marker is also loaded in one of the wells in order to determine the molecular weight of other proteins. After that, the samples are affixed to the remaining wells.
4. After the loading, the current is passed across the gel. The proteins which are tightly bounded to the SDS will move towards the positive pole because they are negatively charged. The migration of protein is inversely proportional to its size.
5. Next, the gel is set upon a membrane, and current is given across the gel so that all the proteins are transported onto the membrane.
6. Then the Immunoblotting is performed. During this method, the membrane is blocked with non-specific protein to inhibit the antibody from binding to the membrane where the protein absent.
7. The primary antibodies are added they will recognize a specific amino acid sequence. Then the membrane is washed off to remove the unbound primary antibody.
8. Now enzyme-labeled secondary antibodies are added which will recognize the primary antibody. After that, the membrane is washed again to remove the unbounded secondary antibodies.
9. The chemiluminescent substrates are used for identification. The light is being emitted once the substrate has been added and can be detected with film imager.

Western blotting Application

Western blotting has several applications in molecular biology, including:

1. Protein detection: Western blotting is commonly used to detect specific proteins in a sample. By using antibodies that are specific to the target protein, researchers can identify the presence and abundance of the protein of interest.
2. Protein quantification: Western blotting can be used to quantify the amount of a specific protein in a sample. By comparing the signal intensity of the target protein to a known standard, researchers can determine the amount of protein present in the sample.
3. Protein expression analysis: Western blotting can be used to study the expression of specific proteins in different tissues or under different conditions. By comparing the protein levels in different samples, researchers can identify differences in protein expression and gain insights into the regulation of protein expression.
4. Post-translational modification analysis: Western blotting can be used to study post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or acetylation. By using antibodies specific to the modified form of the protein, researchers can identify the presence and abundance of the modified protein and study its potential role in disease.

Overall, Western blotting is a valuable tool in molecular biology that has a wide range of applications in protein detection, quantification, expression analysis, and PTM analysis.

3. Northern blotting

This blotting technique was given by Alwine and used for the detection and analysis of RNA in a given sample. The Northern blotting is completed in the following steps;

1. First of all extract and purify mRNA from the cells.
2. After that separate those purified mRNA on agarose gel containing the formaldehyde which is a denaturing agent for the RNA.
3. Immersed the gel within a depurination buffer for 5-10 minutes and then washed it with water.
4. The RNA fragments are transferred onto the carrier membrane such as aminobenzyloxymethyl filter paper.
5. The UV or heat is applied to fix the RNA onto the membrane.
6. Add DNA labeled probe for hybridization.
7. Wash the membrane to remove the unbound probe.
8. Then, the end mRNA-DNA hybrid are then detected by X-ray film.

Northern blotting Applications

Northern blotting has several applications in molecular biology, including:

1. Gene expression analysis: Northern blotting can be used to study the expression of specific genes in different tissues or under different conditions. By comparing the hybridization patterns of mRNA from different samples, researchers can identify differences in gene expression and gain insights into the regulation of gene expression.
2. Identifying alternatively spliced transcripts: Northern blotting can be used to identify and distinguish between different splice variants of a particular gene. By designing probes that are specific to different splice variants, researchers can determine which variants are present in a particular sample.
3. Detecting gene fusions: Northern blotting can be used to detect gene fusions, which occur when two separate genes are fused together. By designing probes that are specific to the fusion transcript, researchers can identify the presence of the fusion gene and study its potential role in disease.
4. Studying small RNAs: Northern blotting can be used to study the expression and processing of small RNAs, such as microRNAs and siRNAs. By designing probes that are specific to these small RNAs, researchers can identify their expression patterns and gain insights into their regulatory roles.

4. Eastern blotting

This blotting technique was given by Bogdanov and used for the detection and analysis of carbohydrate epitopes including glycoconjugates and lipids. The Eastern blotting is completed in the following steps;

1. The supplied molecules are separated by using gel electrophoresis.
2. These molecules are transferred horizontally on the nitrocellulosic membrane.
3. Add the primary antibody within the solution. These antibodies will recognize a specific amino-acid sequence.
4. Wash the membrane to remove the unbound primary antibody.
5. Add the labelled secondary antibody.
6. These labelled probes confirm the molecule of interest.

Eastern blotting Application

Eastern blotting has several applications in glycoprotein research, including:

1. Glycan profiling: Eastern blotting can be used to identify and profile the glycan structures present on a particular glycoprotein. By using different lectins with specific binding properties, researchers can identify different glycan structures and their relative abundance.
2. Disease diagnosis: Abnormal glycosylation patterns have been associated with many diseases, including cancer and genetic disorders. Eastern blotting can be used to identify changes in glycan structures associated with these diseases and aid in their diagnosis and treatment.
3. Vaccine development: Many vaccines are based on glycoproteins, and the glycan structures on these proteins can influence their immunogenicity and efficacy. Eastern blotting can be used to analyze the glycan structures present on vaccine candidates and optimize their design and development.
4. Quality control: Eastern blotting can be used to monitor the quality and consistency of glycoprotein production, particularly in the pharmaceutical industry. By analyzing the glycan structures present on glycoproteins, researchers can ensure that they meet the necessary quality standards and are safe for use.

Overall, Eastern blotting is a valuable tool in glycoprotein research that has a wide range of applications in glycan profiling, disease diagnosis, vaccine development, and quality control.

There is another type of blotting technique known as Dot Blot or dot blotting. In molecular biology, the dot blot technique is used to detect proteins. It is a simplification of the western blot method. In this method, the sample is directly transferred onto the membrane. The electrophoresis step is absent in dot blotting.

Transfer Methods

There are three different methods by which denatured DNA is transferred to the membrane such as;

• Capillary Transfer: In this method the capillary movement of buffer from the soaked paper to the dry paper helps in the movement of Denatured DNA from the gel to the membrane.
• Electrophoretic Transfer: In this method the Electric current helps in the movement of the DNA from the gel to the membrane.
• Vacuum Transfer: In this method the force from suction helps to moves the DNA from the gel to the membrane.

Advantages of Blotting Techniques

1. High sensitivity: Blotting techniques can detect small amounts of nucleic acids or proteins, making them highly sensitive and useful for detecting rare or low-abundance molecules.
2. Specificity: Blotting techniques can be designed to detect specific molecules, such as a particular gene or protein, making them highly specific and useful for identifying and characterizing specific molecules in complex samples.
3. Quantitative: Blotting techniques can be used to quantify the amount of a particular molecule in a sample, making them useful for studying changes in gene or protein expression over time or under different conditions.
4. Flexibility: Blotting techniques can be adapted to a wide range of molecular biology applications, from gene expression analysis to protein detection and post-translational modification analysis.

Disadvantages of Blotting Techniques

1. Time-consuming: Blotting techniques can be time-consuming, involving multiple steps and long incubation times, making them unsuitable for rapid or high-throughput analysis.
2. Technical skill: Blotting techniques require technical expertise and specialized equipment, making them difficult for inexperienced researchers to perform.
3. Cost: Blotting techniques can be expensive, requiring expensive reagents and specialized equipment, making them less accessible to researchers with limited resources.
4. Limited throughput: Blotting techniques are typically performed on a small number of samples at a time, making them less suitable for large-scale studies or screening applications.

In summary, while blotting techniques have many advantages and are essential tools in molecular biology research, they also have limitations that need to be considered when deciding on the appropriate method for a particular application.

FAQ

References

• https://www.rroij.com/open-access/types-of-blotting-.php?aid=79704#19
• https://www.web-books.com/MoBio/Free/Ch9D.htm
• https://www.slideshare.net/Chepkitwai/blotting-techniques-61293002
• https://en.wikipedia.org/wiki/Dot_blot