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 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.
There are mainly four types of blotting such as;
- Southern blotting
- Western blotting
- Northern blotting
- Eastern 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;
- First of all the Large DNA is fragmented into small pieces by usign Restriction endonucleases.
- This fragmented DNA are separated according to their size by using gel electrophoresis.
- The Larger DNA fragments are treated with HCl, which causes depurination of DNA fragments.
- 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.
- The membrane is exposed to ultraviolet radiation as result the fragments weill permanently attached to the membrane.
- 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.
- The membrane is washed using an SSC buffer to remove the excess probe.
- Finally the membrane is visualized on X-ray film with the help of autoradiography.
Southern blotting Applications
- Used in RFLP (Restriction fragment length polymorphism) mapping.
- Southern blotting used in phylogenetic analysis.
- It is used to detect gene rearrangements.
Southern Blotting is a molecular biology technique used for the identification of a specific DNA sequence in a collected DNA sample.microbiologynote.com
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;
- First of all, the desired proteins are isolated from the particular sample.
- The beta- mercaptoethanol (BME) and Sodium dodecyl Sulfate (SDS) is added to the protein suspension.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- Used for different clinical purposes.
- Used to detect a specific protein in a very low quantity.
- Used to quantifying a gene product.
In molecular biology Western blotting is a rapid and sensitive assay for detection and characterization of proteins. This technique exploits themicrobiologynote.com
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;
- First of all extract and purify mRNA from the cells.
- After that separate those purified mRNA on agarose gel containing the formaldehyde which is a denaturing agent for the RNA.
- Immersed the gel within a depurination buffer for 5-10 minutes and then washed it with water.
- The RNA fragments are transferred onto the carrier membrane such as aminobenzyloxymethyl filter paper.
- The UV or heat is applied to fix the RNA onto the membrane.
- Add DNA labeled probe for hybridization.
- Wash the membrane to remove the unbound probe.
- Then, the end mRNA-DNA hybrid are then detected by X-ray film.
Northern blotting Applications
- Used for screening.
Northern blotting or Northern hybridization is a widely used technique in molecular biology to determine the molecular weight of mRNA and tothemicrobiologynote.com
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;
- The supplied molecules are separated by using gel electrophoresis.
- These molecules are transferred horizontally on the nitrocellulosic membrane.
- Add the primary antibody within the solution. These antibodies will recognize a specific amino-acid sequence.
- Wash the membrane to remove the unbound primary antibody.
- Add the labelled secondary antibody.
- These labelled probes confirm the molecule of interest.
Eastern blotting Application
- Used to detect protein modification.
- Eastern blotting used for binding studies by using various ligands.
- Eastern blotting used to purify various phospholipids.
A Molecular Biology Technique is used for the identification of Post Translation Modification mostly carbohydrate Epitopes by transferring onto a membrane andmicrobiologynote.com
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.
Dot Blot is a simplified technique of western blotting, which is mainly used for the detection of proteins.microbiologynote.com
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.