Western Blotting Technique, is the technique of molecular that detect specific proteins and extract in a tissue which are followed by gel electrophoresis. Similarly Northern Blotting is the technique which dtudy about the gene expression which is helped by the RNA while on the otherhand, Southern blotting which detect the specific DNA in a blood or tissue sample.
Western Blotting Technique | Northern Blotting, Southern Blotting
Introduction of Blotting
It is a common technique that is used in the field of molecular biology. Blots are techniques for transferring DNA, RNA, and proteins onto a carrier so that it can be separated and often follows the use of gel electrophoresis. There are different types of blotting such as Southern Blotting, Western Blotting Technique, northern blotting, and eastern blot. The southern blot is used for transferring DNA, the north blot for RNA, the western blot for protein, and the eastern blot for lipids. By using a probe we have to detect the interest of molecule.
The Blotting of nucleic acid is the central technique for hybridization studies. Nucleic acid labeling and hybridization on membranes have formed the basis for a range of experimental techniques involving an understanding of gene expression, organization, etc.
Identifying and measuring specific proteins in complex biological mixtures, such as blood, have long been important goals in scientific and diagnostic practice. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and genetic counseling.
This process can be done after the gel electrophoresis, by transferring the molecules from the gel onto the surface of the blotting membrane. We can visualize these transferring molecules by using staining. The latter is done with antibodies that bind only to some molecules of the blot after proper washing, this activity is visualized by incubation with proper reactive, rendering a color deposit on the blot which is registered by photographic film.
Types of blotting.
- Southern blotting
- Western blotting
- far-western blot
- Southwestern blot
- Eastern blotting
- Far-eastern blot
- Northern blotting
- Reverse northern blot
- Dot blot
List of Blots | Western Blotting Principle Procedure And Application
- Southern blot for DNA
- Northern blot for RNA
- Eastern blot for post-translational modification
- Dot blot
- Western blot for proteins
- Far-eastern blot for glycolipids
- far-western blot for protein-protein interactions
- Reverse northern blot for RNA
Southern blotting Uses:
A southern blotting is a routine method that is used in molecular biology for the detection of a specific DNA sequence in DNA samples. It is named after Edward M. Southern. It combines the transfer of electrophoresis- separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. It involves some steps.
- Cut the large weighted DNA into small fragments by using restrictions endonucleases
- If you want to perform a southern blot, you would first want to separate DNA based upon size in a gel along an electric field. These fragments are electrophoresis on separating gel so that they can separate according to their size.
- If DNA fragments are much larger in size and your larger fragments, firstly the gel should be treated with HCI, cause depurination of DNA fragments, at the top your smaller fragments are going to be at the bottom.
- When you’re done running your gel, then transfer that to a membrane. After separating these fragments, nitrocellulose sheet over the separating gel. Apply pressure over the membrane so that proper interaction can occur between these two. So it’s like making a sandwich: gel, membrane on top, a stack of paper towels.
- Then the membrane is exposed to ultraviolet radiation so that the fragments are permanently attached to the membrane and you’re going to allow a solution to pass through the gel up to the membrane.
- After that 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.
- After the hybridization process, the excess probe is washed away and it can be visualized on X-ray film with the help of autoradiography, up to the membrane it is going to be a soft gradient that pushes it through.
Principle of southern blotting
- The molecules are immobilized on a matrix.
- Any unbound probes are then removed
- The mixture of molecules is separated
- The place where the probe is connected corresponds to the location of the immobilized target molecule.
- The probe is added to the matrix to bind to the molecules.
- Application of southern blotting.
- Southern blots are used in gene discovery, mapping, evolution and development studies, diagnostics, and forensics.
- Southern blot is used to detect the presence of a particular bit of DNA in a sample.
- Southern blots allow investigators to determine the molecular weight of restriction fragment and to measure relative amounts in different samples.
- In regards to genetically modified organisms. Southern blotting is used for testing to ensure that a particular section of DNA of a known genetic sequence has been successfully incorporated into the genome of the host organisms.
- Analyse restrictions digestion fragmentation of DNA or a biological sample.
- Analyze the genetic patterns which appear in a person’s DNA.
western blot gel electrophoresis
A western blotting technique is used for the detection of specific proteins in complex samples. Western blotting is named after W. Neal brunette. This method is used for the detection and analysis of protein. Proteins are first separated by size using electrophoresis before being transferred to an appropriate blotting matrix.
Far western blot
It is similar to a western blot or Western Blotting Technique, the far-western blot is used protein-protein interactions to detect the presence of a specific protein immobilized on a blotting matrix.it is used to detect the presence of the protein-protein complex, making the far western blot a specific case of the western blot.
Southwestern blotting Technique
It is based on the southern blot and used to identify DNA binding proteins by their ability. The proteins are separated by gel electrophoresis and are subsequently transferred to nitrocellulose.
It involves the following steps.
- Isolating the protein from a particular sample. After that beta-mercaptoethanol (BME) and sodium dodecyl sulfate (SDS) are added to the protein suspension. Determine the protein concentration of each sample with a protein quantification assay.
- After that protein – the SDS complex is placed on top of the gel in the well. some samples may need to be reduced. this is achieved by boiling some samples in a buffer, load samples along with a molecular weight marker. A molecular weight marker is also loaded in one of the wells to determine the molecular weight of other proteins. Run the gel in a running buffer
- Once the samples and markers are loaded then current is passed across the gel following electrophoresis, assemble the transfer unit, including the gel. PVDF and filter paper. After that protein is pulled down to the positive pole of the well because it is tightly bound to SDS which is negatively charged.
- Then, transfer the proteins to the membrane with transfer buffer and placed a gel against a membrane and current is passed across the gel so that all the proteins are transferred onto the membrane
- When the immune logging is done. Prepare the blocking buffer and incubate the membrane to reduce nonspecific binding to prevent the antibody from binding to the membrane where the protein is not present.
- Wash the membrane in TSBT, and incubate with conjugated secondary antibody diluted in blocking buffer. these antibodies are responsible for recognizing a specific amino acid sequence. Then wash it to remove unbound primary antibody and add secondary antibody.
- The light is being emitted once the substrate has been added and can be detected with a film imager. Follow manufacturer dimensions for chemiluminescent detection.
Western Blotting Principle Procedure And Application
- A western blotting technique is also used as the definitive test for bovine spongiform encephalopathy.
- Some forms of Lyme disease testing employ western blotting.
- The confirmatory HIV test employs a western blot to detect anti-HIV antibodies on a human serum sample. Proteins from known HIV infected cells are separated and blotted on a membrane then, the serum to be tested is applied in the primary antibody incubation step, a free antibody is washed away, and a secondary anti-human antibody linked to an Andy enzyme signal is added.
The eastern blot is used for the detection of specific posttranslational modification of proteins. eastern blotting is given by Bogdanov. This method is used to identify carbohydrate epitomes including glycoconjugates and lipids. Proteins are separated by gel electrophoresis before being transferred to blotting. Mostly blotted proteins after transferring onto the membrane are analyzed for PTMs by using a probe and hence identify carbohydrates and lipids. Whereupon post-traditional modifications are detected by specific substrates or antibodies. It involves the following steps.
- Firstly, separate the targeting molecules vertically by using gel electrophoresis.
- Then, transferred separated molecules horizontally on the nitrocellulosic membrane.
- After that add the primary antibody to a solution. These antibodies are responsible for recognizing an amino acid sequence. Then Properly wash it to remove unbound primary antibody and add labeled secondary antibody.
- This labeled probe will confirm the molecule of interest.
Check Other Post– Simple Real-Time Polymerase Chain Reaction
Far eastern blot:
The far eastern blot is for the detection of lipid-linked oligosaccharides. It is first used to separate the lipids by physical and chemical character, then transferred to a blotting matrix before the oligosaccharides are detected by specific binding proteins.
The northern blot is for the detection of specific RNA sequences in complex samples. The northern blotting first separated samples by size via gel electrophoresis before they are transferred to a blotting matrix and detected and labeled RNA probes. Northern blotting is given by Alwine. The method is used to analyze and detection of RNA in a sample. It involves some steps
- Firstly, purify RNA and extract from the cells and isolate the RNA.
- Prepare the gel and insert the gel tray into the apparatus. Fill with MOPS buffer, load the samples, and include a molecular weight marker. Run the gel, then trim the gel before blotting. Separate these RNAon agarose gels containing formaldehyde as a denature get agent for the RNA.
- This gel is immersed in a depurination buffer for 5-10 minutes and wash the gel in SSC, then assemble the transfer unit with the gel, filter paper, and nylon membrane. When the transfer is complete, place the membrane in a UV-cross-linker.
- Add DNA labeled probe for hybridization and incubate the blot with the probe.
- Following incubation, perform a low stringency wash off the unbound probe, and at the end MRNA- DNA hybrid is then detected by X-Ray film.
- Discard antibody solution, wash the membrane, and follow manufacturer directions for chemiluminescent detection.
Application Northern blotting
- Detection of mRNA transcript size.
- A standard for the direct study of gene expression at the level of mRNA.
- Study RNA degradation.
- Study RNA half-life.
- Study IRES-to remove the possibility of RNA digestion vs. 2nd cistron translation.
- Often used to confirm and check transgenic.
- Study RNA splic8ng – can detect alternatively spliced transcripts.
Disadvantage of Northern blotting.
- The whole process of northern blotting takes a long time usually, from sample preparation to nonradioactive detection.
- Often radioactive is used. This prevents ease of performing it, use, and disposal. New methods of nonradioactive detection have been generated allowing nonradioactive detection.
- The standard northern blot method is relatively less sensitive than nuclear protection assays and RT-RCR.
- IF RNA samples are even slightly degraded by RNases, the quality of the data and quantization of expression are quite negatively affected.
- Detection with multiple probes is a problem. Often, the membranes must be stripped before hybridization and detection with a second probe.
- The sensitivity of northern blots may be increased with the use of nylon positively charged membranes, use of a highly specific anti-sense probe.
Reverse northern blotting:
The reverse northern differs from both northern and southern blot in that DNA is first immobilized on a blotting matrix and specific sequences are detected with labeled RNA probes.
It is a special case of any of the above blots where the analysis is added to the blotting matrix as opposed to separating the sample by electrophoresis before blotting.
Frequently asked questions.
- What is the blotting technique?
Ans- it is a technique for transferring DNA, RNA , and proteins onto a carrier so they can be separated by using gel electrophoresis. The southern used in transferring DNA, the northern blot for RNA, and the western blot for protein.
- What is blotting and its types?
Ans- it is a common technique that is widely used in the field of molecular biology. Their types are southern, western, northern, and eastern are applicable for different types of macromolecules like lipids, RNA, DNA, and proteins.
- What is blotting face?
Ans- Blotting sheets work their magic by soaking up excess sebum on the skin, subsequently making it look and feel more matte much like powder or any other oil- sopping product on the market.
- How do blotting papers work?
Ans- It soaks up the excess shine on the surface of your skin while targeting oily sebum deep in your follicles. Pressing a blotting paper into your skin to absorb oil does remove some shine at a cost. The most important thing to do when you’re struggling with oily skin is to identify the cause and to solve it.
- Why western blotting technique is used?
Ans- western blots or Western Blotting Technique can be used to evaluate the size of a protein of interest and to measure the amount of protein expression. It is a laboratory method used to detect specific protein molecules from among a mixture of proteins.
- Is blotting paper bad for skin?
Ans- oil blotting sheets, along with the other unnecessary oils, which actually prompts the skin to increase oil production to compensate. So the answer is yes
- Who discovered blotting techniques?
Ans- the scientists named this ethic as northern blot. A doc who is working in the lab of Robert nowiski at Fred Hutchinson cancer research center USA W Neal Burnett’s identify specific antigens in a protein mixture.
- Do oil blotting sheets help acne?
Ans- the cause of acne is sebum which causes pores and bacterial growth. Blotting does not reduce acne and though blotting paper can’t reduce acne. There is still has a place in your makeup and gym bag. It is useful if your goals are to avoid powder makeup.
- How do you blot your face?
Ans-blotting paper is the perfect solution. blotting paper is so smooth which removes oil without taking off your makeup. It is the most perfect and quick solution for any oily face, just take out a sheet and blot all of your faces with oily spots.
Now its time to end this article, I hope this article would be helpful for you. Different blotting is used to detect different macromolecules such as southern blotting is used for DNA whereas another’s blotting is for, western blotting tehnique is for protein analysis and eastern is for proteins and carbohydrates. This article is focused on different techniques and applications used in particular blotting.
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