Definition of blotting: Blotting means the transfer of sample that is obtained biologically from the gel to the membrane.
Definition of western blotting: It is the technique by which the identification of particular protein from the sample is done. It is also called protein blotting. It takes place by the interaction of antibody with the antigen. The key method used in the process of western blotting is the use of primary and secondary antibodies to find the target protein.
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Western blotting procedure: Western blotting is completed using different steps. The steps used are as follows:
- Firstly, separation of macromolecules is done. This process is completed using electrophoresis. In the process of electrophoresis, molecules are separated by their size. Smaller and lighter molecules travel faster and larger molecules remains behind. The complete process is carried under the influence of electric current.
- Once the molecules are separated, then the transfer of molecule in a membrane is done. It can be either nitrocellulose paper or nylon sheath.
- Then the membrane is blocked so that antibodies bind only to the target protein. Blocking of membrane is done using BSA and TBS for the non- specific binding of antibodies.
- Primary antibody is then added and the mixture of antibody and membrane is incubated. It can be done from 30minutes to overnight.
- After the incubation process is over, membrane is washed for any unbound antibody and then secondary antibody is again added.
- It is again incubated in the room temperature for 2-3 hours and then TBST is added, and hence the resultant is visualized by different means either by the use of fluorescent process, radioactive detection etc.
Image Reference: http://commons.wikimedia.org/wiki/File:Western_blotting.png
Application of western blotting:
- Western blotting is used for identification of different diseases like that of hepatitis B, BSE, HIV etc.
- It is used in scientific research.
- It is used to determine the expression of target protein
Southern blotting: It is the technique used for the identification of particular sequence of DNA.
Southern blotting procedure: The procedure of southern blot involves the following steps:
- Since DNA is a long strand, hence it is important to break it into pieces for the process to carry on. Thus, it is broke into pieces by the use of restriction enzyme. There are different types of restriction enzymes that are used to obtain the fragment of DNA.
- After DNA is fragmented into smaller pieces, then electrophoresis is done in order to separate the fragments of DNA according to their size. The smaller fragments run faster and the larger fragments are left behind.
- Now the separated DNA fragments are transferred from gel to the membrane called nylon sheath or nitrocellulose paper. Hence, this maintains the pattern of gel without changing the pattern of transfer.
- The specific probe is then used and incubated with the membrane.
- This probe is radioactively labeled and will base pair with the DNA segment and hence when visualized under an X-ray film, will help to find out about the fragment of interest.
Image Reference: http://commons.wikimedia.org/wiki/File:Southern-Blot-Autoradiogramm.jpg
Uses of southern blotting: There are different uses of southern blotting. Some of them are:
- It is used to detect specific DNA molecule.
- It is used in DNA Fingerprinting
- It is used in cloning and many other scientific methods.
Definition of northern blotting: It is the technique that is used to find the sequence of RNA/ mRNA from the given sample.
Northern blotting procedure: Northern blotting also involves the process like that of southern blotting but the difference is the involvement of RNA rather than DNA. Hence, the process is described below:
- Firstly, RNA is separated using the process of gel electrophoresis.
- Then it is transferred to the sheet of nylon paper or the nitrocellulose paper.
- Now a radiolabelled DNA probe is used that bind complimentary with the RNA molecule. Use of substrate helps to detect the required RNA sequence under the x-ray film.
Image Reference: http://commons.wikimedia.org/wiki/File:Northern_Blot_Scheme.PNG
- Northern blotting is used to observe the pattern of gene expression.
- These blotting techniques are useful in different fields of scientific field for different researches and experiments.
All these three kinds of blotting technique are basically the same. It’s just the difference in the enzyme that is used and some steps that have either been added or deleted.
The various techniques through which it is achieved are discussed in brief as follows:
- Mass Spectrometry
- Isoelectric Focussing
- Scatchard Analysis