Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow researchers to identify specific molecules like DNA, RNA, and proteins that have been separated in a gel. The process involves transferring molecules from a gel onto a membrane, where they are immobilized. Probes are then used to detect and visualize the target molecules on the membrane. These techniques have various applications, including DNA analysis, gene expression studies, disease diagnosis, and protein analysis. However, non-specific binding can sometimes generate false positives that techniques aim to reduce.

1. Blots

2. INTRODUCTION
● Blotting provides a means of identifying specific molecules out of
a mixture
● Blotting is technique in which nucleic acids i.e., RNA and DNA or
proteins are transferred onto a specific membrane (nitrocellulose
PVDF or nylon membrane).
● This process can be done just after the gel electrophoresis, by
transferring the molecules from the gel onto the surface of
blotting membrane.
● One can visualize these transferring molecules by using staining.
Examples: Ethidium bromide, Crystal violet, Safranin and
Osmium tetroxide etc
● Blot is an imprint of the bands of nucleic acid or protein on a
membrane, that were in the gel
.

3. 1. Electrophoretic separation of protein or of nucleic acid fragments in the sample
2. Transfer to and immobilization on membrane
3. Binding of analytical probe to target molecule on membrane
4. Visualization of bound probe
Molecules in a sample are first separated by electrophoresis
An agarose electrophoresis gel, containing the fractionated restriction fragments,is. placed on a
filter paper wick that forms a connection between the gel and a reservoir of high-salt buffer. The
nitrocellulose membrane is placed on top of the gel and covered with a tower of paper towels
that are held in place with a weight. Capillary action results in the buffer soaking through the filter
paper wick, gel and membrane and in to the paper towels.As the buffer passes through the gel
the fragments are carried with it into the membrane,where they become bound to the
nitrocellulose. This immobilizes the protein or DNA fragments, thus provides a faithful replica of
the original separation, and facilitates subsequent biochemical analysis.
After being transferred to the support medium the immobilized protein or nucleic acid fragment is
localized by the use of probes, such as antibodies or DNA, that specifically bind to the molecule
of interest.
Finally, the position of the probe that is bound to the immobilized target molecule is visualized
usually by autoradiography.

4. Southern Blot
● Southern blot is the process of transfer of DNA fragments that are separated by
electrophoresis onto a membrane for immobilization and identification.
● The most basic form of the technique is used to determine the size of a DNA
fragment from a complex mixture of genomic DNA.
● The technique is also relatively quantitative and can be used to determine the
number of copies of a segment present in a genome.
● Southern Blotting can be modified based on the choices of the membrane, transfer
buffer, and method. The most commonly used membrane is the nitrocellulose
membrane, as it is robust and can be reprobed a number of times.
● The technique was discovered by Edwin Southern, and the technique was named
after him. The technique later gave rise to other techniques like Western Blotting and
Northern Blotting that are based on the same principle.

5. Principle of Southern Blot
● The principle of southern blotting is similar to the blotting technique involving the
transfer of biomolecules from a membrane to another for detection and identification.
● The DNA to be analyzed is digested with restriction enzymes and fractionated by size
by the process of agarose gel electrophoresis.
● The DNA strands are denatured by alkaline treatment and are transferred to a nylon or
nitrocellulose membrane by the blotting process.
● The strands on the membrane are immobilized on the surface by baking or UV
irradiation. The DNA sequences on the membrane can be detected by the process of
hybridization.
● Hybridization reactions are specific as the probes used bind to target fragments
consisting of complementary sequences.
● The probes used are labeled with different components that can be visualized by
different methods depending on the type of probes used.

6. Northern Blot
● Northern blot is a technique based on the principle of blotting for the analysis of
specific RNA in a complex mixture.
● The technique is a modified version of the Southern Blotting
● The principle is identical to southern blotting except for the probes used for the
detection as northern blotting detects RNA sequences.
● This technique provides information about the length of the RNA sequences and the
presence of variations in the sequence and has also been used for the quantification of
RNA sequences.

7. Principle of Northern Blot
● The principle of the northern blot is the same as all other blotting technique that is
based on the transfer of biomolecules from one membrane to another.
● The RNA samples are separated according to their size by gel electrophoresis. Since
RNAs are single-stranded, these can form secondary structures by intermolecular base
pairing and thus separation of the RNA segments is performed under denaturing
conditions.
● The separated RNA fragments are then transferred to a nylon membrane.
● The transferred segments are immobilized onto the membrane by fixing agents. The
RNA fragments on the membrane are detected by the addition of a labeled probe
complementary to the RNA sequences present on the membrane.
● The hybridization detects the RNA as the specificity of hybridization between the
probe, and the RNA allows the accurate identification of the segments.
● Northern blot utilizes size-dependent separation of RNA segments and thus can be
used to determine the sizes of the transcripts.

8. Western Blotting
Western blotting is named after W. Neal Burnette. This method is used for detection
and analysis of protein in a given sample It involves the following steps
● Firstly, isolating the protein from particular sample.
● After that beta- mercaptoethanol (BME) and Sodium dodecyl Sulfate (SDS) is
added into the protein suspension
● Then, protein- SDS complex is placed on top of the gel in the well. A molecular
weight marker is also loaded in one of the well in order to determine the
molecular weight of other proteins. After that the samples are added in the
remaining wells
● Once the samples and markers are loaded then current is passed across the gel.
Protein is pulled down to the positive pole of the well because it is tightly bound to
SDS which is negatively charged. Movement of protein is inversely proportional to
its size

9. Eastern Blotting
● This method is used to identify carbohydrate epitopes including glycoconjugates
and lipids .Mostly blotted proteins after transferring onto the membrane are
analyzed by using a probe and hence identify carbohydrates and lipids. It involves
the following steps:
● Firstly, targeted molecules are vertically separated by using gel electrophoresis
● .Then, these separated molecules are transferred horizontally on the nitrocellulosic
membrane After that primary antibody is added to the solution. These antibodies
are responsible for recognizing a specific amino-acid sequence. Then wash it to
remove unbound primary antibody and add labelled secondary antibody

10. ● After this step, gel is placed against a membrane and current is passed across
the gel so that all the proteins are transferred onto the membrane
● Then Immunoblotting has to be done. In this method, firstly block the
membrane with non-specific protein in order to prevent antibody from
binding to the membrane where the protein is not present
● After that primary antibody is added to the solution. These antibodies are
responsible for recognizing a specific amino-acid sequence. Then wash it to
remove unbound primary antibody and add secondary antibody Now these
antibodies are conjugated with an enzyme and recognize the primary antibody.
● Lastly, another wash is done to remove unbound secondary antibody Here,
chemiluminescent substrate is used for detection. The light is being emitted
once the substrate has been added and can be detected with film image

11. Application of Northern blotting
● The technique can be used for the identification and separation of RNA fragments
collected from different biological sources.
● Northern blotting is used as a sensitive test for the detection of transcription of DNA
fragments that are to be used as a probe in Southern Blotting.
● It also allows the detection and quantification of specific mRNAs from different
tissues and different living organisms.
● Northern blotting is used as a tool for gene expression studies related to
overexpression of cancer-causing genes, and gene expression during transplant
rejects.
● Northern blotting has been used as a molecular tool for the diagnosis of diseases like
Crohn’s disease.
● The process is used as a method for the detection of viral microRNAs that play
important roles in viral infection.

12. Application of Southern blotting
● Southern blotting has many applications in the field of gene discovery, mapping,
evolution, and diagnostic studies.
● The technique can be used for DNA analysis to detect point mutations and other
structural rearrangements in the DNA sequences.
● The method also allows the determination of molecular weights of the restriction
fragments, which helps in the analysis of such fragments.
● Since the technique enables the detection of a particular DNA segment, it can be
used in personal identification via fingerprinting.
● It can be used in disease diagnosis as well as prenatal diagnosis of genetic diseases.

13. Applications of Western blot
● Western blotting is an excellent method with high sensitivity in order to detect a
particular protein even in low quantity.
● Western blotting has been used in the clinical diagnosis of different diseases. The
confirmatory test for HIV involves a western blot by detecting anti-HIV
antibodies in the serum.
● The technique has been used to quantify proteins and other gene products in
gene expression studies.
● Since western blotting detects the proteins by their size and ability to bind to the
antibody, it is appropriate for evaluating the protein expressions in cells and
further analysis of protein fractions during protein purification.
● Western blotting is also used for the analysis of different biomarkers like growth
factors, cytokines, and hormones.

14. Applications of Eastern blot
● The most important application of eastern blotting is the analysis of post-translational
modifications in proteins.
● The technique has been used to identify and purify different plant products.
● Eastern blotting also allows the detection of modifications in proteins of different origins.
● It also helps to study the nature of interactions between different molecules by the use of
ligands.
● Eastern blotting has been extensively used to compare modifications in proteins obtained
from different bacterial species.
● It is also used to detect carbohydrate epitopes in different proteins.

15. PROBLEMS
● Nonspecific interactions between blotted proteins and unrelated secondary antibodies
generate false positives in immunoblotting techniques.
– Some procedures have been developed to reduce this non specific interaction , but they
may work in specific applications and be ineffective in others.
– However, the procedures makes it usable in other applications that come up against this
kind of problems

16. References
https://bio.libretexts.org/Bookshelves/Biochemistry/Book%3A_Biochemistry_Fre
e_and_Easy_(Ahern_and_Rajagopal)/09%3A_Techniques/9.11%3A_Blotting
Ghosh, R., Gilda, J. E., & Gomes, A. V. (2014). The necessity of and strategies for
improving confidence in the accuracy of western blots. Expert review of
proteomics, 11(5), 549-560
.https://www.researchgate.net/publication/20678377_Blotting_techniques_for_th
e_study_of_DNA_RNA_and_proteins

17. References
https://microbenotes.com/northern-blot/
https://microbenotes.com/southern-blot/
https://microbenotes.com

18. THANK YOU