The document discusses various blotting techniques used to transfer and detect DNA, RNA, and proteins, including Southern blotting, Northern blotting, and Western blotting. Southern blotting was developed by Edwin Southern in 1975 to detect specific DNA sequences and involves transferring DNA from a gel to a membrane. Northern blotting, developed in 1979, detects specific RNA sequences and involves transferring RNA. Western blotting, developed in 1981, detects specific proteins and involves separating proteins by gel electrophoresis, transferring them to a membrane, and using antibodies to detect proteins of interest. These techniques are widely used analytical tools.

1. Blotting techniques
Gurvinder kaur
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GADVASU

2. What is blotting?
➢ Blots are techniques for transferring DNA , RNA and proteins onto a
carrier so they can be separated, and often follows the use of a gel
electrophoresis.
➢ Blotting technique are widely used analytical tools for the specific
identification of desired DNA/RNA fragments from thousands of
molecules.
➢ Process of immobilization of sample nucleotides on solid support
(nitrocellulose or nylon membrane)

3. Types of Blotting Techniques
Blotting
techniques
Southern
Blot
Northern
Blot
Western
Blot

4. Types of Blotting Techniques

5. Southern Blotting
➢ SOUTHERN BLOTTING Professor Sir Edwin Southern, Professor of
Biochemistry and Fellow of Trinity developed this method in 1975.
➢ Southern won the Lasker Award for Clinical Medical Research prize for
the method of finding specific DNA sequences.
➢ He developed this procedure at Edinburgh University more than 30 years
ago.
➢ The technique is known as DNA transfer or 'Southern blotting'
Professor Sir Edwin Southern

6. Southern Blotting-Principle
In conjuction with filter hybridization, this method relied on
the complimentary nature of double-stranded DNA and its
reversible denaturation/renaturation properties to detect the
presence and abundance of target DNA sequences in the
fractionated DNA population using a labeled probe of the
DNA of interest.

7. Southern Blotting

8. Southern Blotting
• Three methods are available for transfer of fractionated DNA from an
agarose gel onto a solid support.
1. Capillary Transfer method- conventional transfer method , relies on a gel
sandwich set up, where a gel is placed over a buffer-soaked wick and
overlaid with a piece of transfer membrane.

9. Scheme of capillary transfer methodfor Southern blotting

10. 2. Alkaline Transfer method – developed by Reed and Mann in 1985. It
takes the advantage of the positively nylon membrane to covalently bind
negatively charged nucleic acids during the capillary transfer. With the
alkaline transfer buffer , DNA is transferred in a fully denatured state with
a rate faster than that of high- salt buffer method. (2h vs. overnight).

11. Procedure:
▪ Preheat the gel with depurination and denaturation solutions
▪ Set up transfer apparatus, using a positively charged nylon membrane and
0.4 N NaOH as transfer buffer instead of 10X SSC.
▪ Allow the transfer to occur for 2h to overnight.
▪ Disassemble the setup, rinse the membrane with 2X SSC and air dry. The
membrane need not be UV cross linked. It can be stored or used for
hybridization immediately.

12. 3. Vacuum Transfer Method- efficient vacuum-based transfer method that
utilizes commercially available vacuum blotters.
▪ Streamlined gel treatment and DNA transfer can be completed in about an
hour.
▪ Cut a nylon membrane to the size of the gel,, and cut a silicone mask with a
opening smaller than the size of the gel. Masks can be used multiple times.
▪ Place a piece of 3MM Whatmanfilter on the metal grid of the vacublot a
pparatus and wet it distilled water.
▪ Place the cover of the vacublot into the apparatus to hold the mask in place.
▪ Apply the vacuum at 75 mmHg, and leave it on throughout the transfer
process.

13. Applications
• Southern blots are used in gene discovery , mapping, evolution and
development studies, diagnostics and forensics (It is used for DNA
fingerprinting, preparation of RFLP maps)
• It is used for identification of the transferred genes in transgenic
individuals, etc.
• Southern blots allow investigators to determine the molecular weight of a
restriction fragment and to measure relative amounts in different samples.
• Southern blot is used to detect the presence of a particular bit of DNA in a
sample analyze the genetic patterns which appear in a person's DNA.

14. Northern Blotting
➢ Northern blotting is a technique for detection of specific RNA sequences.
➢ Northern blotting was developed by James Alwine and George Stark at
Stanford University (1979) and was named such by analogy to Southern
blotting.
➢ Northern Blot hybridization or RNA blotting is an RNA analysis procedure
in which size and amount of specific mRNA molecules in total RNAs or
poly (A) RNAs can be determined.
➢ RNA molecules are separated according to their sizes by electrophoresis on
a gel under denaturing conditions, transferred to a nitrocellulose or nylon
membrane, and fixed to the membrane.

15. Northern Blotting
➢ The blotted membrane is hybridized with a specific probe, and the mRNA
of interest is then visualized by autoradiography.
➢ The DNA probe is usually stable but needs longer hybridization. The RNA
probe, on the other hand, is much hotter, but is relatively unstable.
➢ Difficulty arises in inactivating the RNase activity.
➢ RNA is single stranded and has a tendency to fold back on itself.

16. Northern Blotting
➢ This allows complementary bases on the same stretch of RNA to base-pair
with each other and form what is termed “secondary structure.”
➢ Because secondary structure can lead to aberrant electrophoretic behavior,
RNA is electrophoretically separated by size in the presence of a
denaturing agent, such as formaldehyde or glyoxal/DMSO
➢ Blot transfer of RNA molecules is carried out by using a chemically
reactive paper prepared by diazotization of aminobenzyloxymethyl to
create diazobenzyloxymethyl (DBM) paper.
➢ The RNA can covalently bind to DBM paper. A good technique for
determining the number of genes (through mRNA) present on a given
DNA.

17. Northern Blotting

18. Applications
• A standard for the study of gene expression at the level of mRNA
(messenger RNA transcripts)
• Detection of mRNA transcript size
• Study RNA degradation
• Study RNA splicing
• Study RNA half-life
• Often used to confirm and check transgenic / knockout mice (animals)

19. Disadvantage of Northern blotting
• Standard northern blot method is relatively less sensitive than nuclease
protection assays and RT-PCR
• Detection with multiple probes is a problem
• If RNA samples are even slightly degraded by RNases , the quality of the
data and quantitation of expression is quite negatively affected.

20. Western blotting
➢ Western blot analysis is based on protein-protein hybridization technique
that is used for immune detection of specific antigen of interest in complex
mixture of proteins.
➢ This is simple sensitive and effective technology used in immunology.
➢ Western blotting Western blotting (1981) is an Immunoblotting technique
which rely on the specificity of binding between a protein of interest and a
probe.

21. Principle of western blotting
➢ A protein mixture is first separated according to molecular size using
sodium dodecyl sulfate –polyacrylamide gel electrophoresis.
➢ The separated protein molecules are then immobilized onto a nitrocellulose
or polyvinylidene difluoride (PVDF) membrane.
➢ The specific protein band of interest is identified by use of a specific
antibody raised against a specific antigen (protein), which can specifically
bind to the antigen (protein) of interest in the protein mixture that is
immobilized on the membrane.

22. • The antigen-antibody complex is then detected by an enzyme linked to a
second antibody and substrate.
• The antigen can also be directly detected with a fluorescence-labeled
antibody, which directly binds to the antigen in a protein mixture.

23. Western blot apparatus

24. Western blotting

25. APPLICATIONS
➢ Western blots are widely used in many types of applications for
confirmatory or quantitative analysis of specific target proteins
➢ Protein band signal confirms protein presence and identity
➢ Position of band informs about protein molecular weight, confirm expected
size
➢ Intensity of band informs about protein amount in the sample

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