Blotting is a method of transferring proteins, DNA, RNA onto a carrier (special membrane). This membrane may be nitrocellulose, PVDF or nylon membrane. This process can be done after gel electrophoresis, by transferring the molecules from gel onto the surface of blotting membrane but sometime samples are directly added onto the membrane.
The process of transferring of the denatured fragments out of the gel and onto a membrane made from nylon (or sometimes nitrocellulose) where they become accessible for analyzing using a probe is blotting. They are analytical techniques used for identification of a specific DNA, RNA or a protein and are collectively referred to as blotting techniques.
The key to this method is hybridization which is a process of forming a double-stranded molecule between a single-stranded probe and a single-stranded target segment. It is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA.
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 understanding of gene expression and its organization, Identifying and measuring specific proteins in complex biological mixtures etc.
Principle of blotting
The mixture of molecules such as proteins, DNA, RNA etc. (Fragmented if necessary) is separated by electrophoresis which are immobilized on a matrix. The probe is added to the matrix to bind to the molecules. Any unbound probes are then removed by washing. The place where probe is bound corresponds to the location of immobilized target molecule.
It involves following steps:
- Nucleic acids, Proteins are extracted and fragmented.
- The denatured fragments are then transferred onto a nylon or nitrocellulose filter membrane which is done by placing the gel on top of a buffer saturated filter paper, then laying nitrocellulose filter membrane on the top of gel and finally placing some dry filter papers on top of this membrane.
- Buffer moves, due to capillary action, from the bottom filter paper through the gel carrying with it the denatured molecules present in the gel.
- Fragmented Molecules becomes attached into nitrocellulose membrane as the buffer passes through it, the process is called blotting and takes several hours or overnight to complete.
The relative positions of bands on membrane remains same as that in gel and there is minimal loss in their resolution (sharpness).
There are basically three types of blotting:
Southern Blot is used to detect DNA, Northern Blot to detect RNA and Western blot is used to detect protein.
However, Eastern blotting is now discovered/developed which is used to identify carbohydrate epitopes including glycoconjugates and lipids.
Blotting techniques are used to identify unique proteins and nucleic acid sequences. They have been developed to be highly specific and sensitive and have become important tools in both molecular biology and clinical research.