BLOTTING

BLOTTING

15.      BLOTTING
Blotting is known as immobilization. Nucleic acid blotting is called as immobilization of nucleic acid (DNA or RNA) on membrane and protein blotting is called as immobilization of protein on the membrane. Blotting is done after the gel electrophoresis, thus transfer of bands of nucleic acid or protein on the membrane is the essential step of blotting.
15.1.    Types of blotting techniques
15.1.1..    Capillary blotting: It is the conventional method of blotting, in this type of blotting, movement of the buffer is carried out through capillary action across the filter paper to the region of low water potential from the region of high water potential. This causes the movement of DNA from the gel onto the membrane, DNA binds to the membrane by ionic interaction because DNA is negatively charged and the membrane is positively charged.


15.1.2.    Electroblotting: In this type of blotting rapid electrophoretic transfer of DNA, RNA and protein take place from gel to membrane in presence high current, thus an external cooling system is also required.

15.1.3.    Vacuum blotting: This type of blotting transfers nucleic acid and protein from gel to membrane. It is very rapid as well there is less chance of diffusion of DNA because the gel treatment can be done in situ, within the vacuum apparatus.


15.2.    Southern blotting
This method is developed by Edwin Southern in 1975 for the transfer of DNA from gel to the membrane. The Procedure starts from the isolation of DNA from the subject, then it gets digested by the restriction enzyme, and the resulting fragment gets separated with the help of agarose gel electrophoresis and a standards DNA leader is also run for determining the size of DNA which gets separated. After that the transfer of DNA from the gel onto the membrane takes place. There is two membrane which is used in Southern blotting. If we use a nitrocellulose membrane, which is an uncharged membrane certain treatment of gel is required for efficient transfer called pre-treatment. Due to pre-treatment larger DNA fragment up to 10 kb can be transferred. Pre-treatment involves depurination followed by alkali treatment. The depurination involves treatment with HCl which in turn cause uniform transfer of DNA fragments having wide size. The alkali treatment causes the hydrolysis of the phosphodiester bond at the depurination site. As a result, DNA becomes single stranded and also become accessible for hybridization with the probe. 

After this step equilibration of gel in neutralizing solution takes place. This pre-treatment step is not required in the case of positively charged nylon membrane which possesses the immense binding capability for nucleic acid as well as high tensile strength. In case of nylon membrane gel is transferred in its native form and then alkali denaturation takes place in situ on the membrane. After the neutralization step, in case of nitrocellulose membrane or just after the electrophoresis DNA on the gel is ready to transfer on the membrane. Neutral transfer buffer is used in case of nitrocellulose membrane and alkaline transfer buffered use in case of nylon membrane to transfer the DNA from gel to membrane. 
At 80°C, vacuum oven baking is used especially for nitrocellulose membrane and it is also used for nylon membrane.  UV cross-linking is also used. In UV cross-linking method, cross-linking occurs between positively charged amino group present on the surface of nylon membrane and the tiny proportion of the thymine residues in the DNA. Subsequently the fixation step, the membrane is allowed to hybridize with the radiolabeled probe (ssDNA or RNA, the probe is oligonucleotide strand which is complementary to our desired sequence whose presence we need to identify in sample bands or sample) by placing the membrane into radiolabelled probe solution. 
Hybridization is performed at low stringent condition (low temperature and high ionic strength) which also allows some non-specific binding along with the maximum rate of hybridization. The unbound probes removed by washing at high stringent condition (high temperature and low ionic strength), thus all nonspecific binding get removed. The region of hybridization gets detected by autoradiography by localizing the membrane in contact of X-ray film and the desired DNA sequence gets identified if present in the sample bands. The size of DNA can be determined related to their position within gel corresponding to the simultaneously run DNA size ladder.
15.2.1.    Application
It is used to study the organization of gene within the genome through mapping the restriction sites in the region of genomic DNA segments. It is also used for mapping of a restriction site in a complex genome, around a single copy gene sequences. It is also used in DNA fingerprinting. By Southern blotting, we can identify homology between two organisms.
15.3.    Northern blotting

This method is developed by Alwine and Stark in 1977 for the transfer of RNA from gel to the membrane. The procedure starts with the isolation of RNA from the cell. The RNA is separated with the help of agarose gel electrophoresis in the presence of the denaturing condition so that inter or intramolecular base pairing does not take place within RNA. Denaturing condition is obtained by the presence of formaldehyde or glyoxal. A standards RNA leader also run for determining the size of RNA which get separated. 
After that the transfer of RNA from the gel onto the membrane takes place. For RNA transfer special type of membrane is used known as diazobenzyloxymethyl (DBM) membrane. DBM membrane is formed by diazotization of aminobenzyloxymethyl paper. RNA binds to membrane covalently when it is transferred to the membrane. After that hybridization of the probe (radiolabeled DNA sequences) takes place. Appropriate nylon membrane and nitrocellulose membrane in appropriate condition can also be used for RNA transfer. Because use of nylon and nitrocellulose convenient then DBM, it suppresses the use of DBM for RNA transfer. The process of probing along with the type of probe, hybridization and post-hybridization are common in northern and southern blotting.
15.4.    Western blotting
Through western blotting, we can identify and quantify the presence of a particular protein (antigen) into a complex mixture of different protein or tissue sample with the help of mAb or polyclonal antibody or primary antibody. In western blotting formation of Ag-Ab complex takes place. The secondary antibody is enzyme-linked. Secondary antibody bind to the primary antibody. A colourless substrate is added which is converted into colour product. As a result identification of particular protein (antigen) occurs. Its principle is same as ELISA. 
Different proteins get separated with the help of SDS-PAGE electrophoresis and protein bands are electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane, nylon or nitrocellulose membrane can also be used. After the transfer of protein to the membrane, the PVDF membrane is blocked by blocking buffer (Bovine serum albumin–BSA or casein or nonfat dried milk) with the addition of Triton X 100. Blocking prevents the nonspecific binding of the antibody to the membrane, thus reduce the noise. After that primary antibody is added and allowed to incubate. As incubation completes, unbound antibody is washed by wash buffer. 
After that, the secondary enzyme-linked antibody is allowed to incubate. As incubation complete unbound secondary antibody is washing by wash buffer then the colourless substrate is added which produce a colour reaction. Identification of protein is done by the standard curve method as described in ELISA. Like ELISA, here also various other methods dire used, like the use of fluorogenic and chemiluminogenic substrates, use of Staphylococcus Aureus protein A- describe in RIA. Another alternative method is the use of biotin attached primary antibody followed by the addition of streptavidin or avidin -conjugate enzyme (HRP enzyme). After that the addition of chromogenic substrate responsible for the detection of Ag-Ab complex.
15.5.    Far Western blotting
It is a variant of western blotting which is used to detect protein-protein interaction, identify ligand-receptor interaction, library screening for interacting protein. Here protein is maintained in its native functional form and is separated with the native page and binds with prey like western blotting transfer to membrane then bait tagged enzyme-linked antibody is added and allowed to incubate. After incubation wash with washing buffer, this result is the removal of unbound antibody i.e. the antibody in which bait not able to bind with prey. 
After that colourless chromogenic substrate is added. If a coloured product is formed then it shows that there is the interaction between bait and prey. If the coloured product is not formed means that there is no interaction between bait and prey.
15.6.    Eastern blotting
Eastern blotting is used to identify the effect of the post-translational modification on protein expression. It is also a variant of western blotting.
15.7.    Zoo blotting
The Comparision of proteins between different animal phylum is done with the help of zoo blot. The comparative study between plant phylum done with the help of Garden blot.


15.8.    Southwestern blotting
This includes the property of both southern and western blotting. It is used to study the interaction between DNA and protein. The Clone which is responsible to express sequence-specific DNA binding protein is isolated with the help of this blotting technique.


Next Previous