he given ppt contains all the blotting techniques which is being studied by students in Biotechnology related subject and this PPT contais all blotting techniques in a very elaborative concise manner includes procedure principle application etc so which itwould help any bio student to take proper knowledge in this topic. I hope you will enjoy the content of the topic and would be able to grasp the topic properly

1. BLOTTING TECHNIQUES
B.Sc (Life Science) 3rd Year
:
Rohit Mondal
B.Sc(Life Science) 3rd yr
Sri Aurobindo College
DSE-Plant Analytical technique

2. What is a blot ?
 In molecular biology blots is a technique for
transferring DNA , RNA and proteins onto a
carrier so they can be separated, and often
follows the use of gel electrophoresis.
Types Of Bloting :
 Southern Blotting: Used For DNA Detection
in a sample
 Northern Blotting : Used For RNA Detection
in a sample
 Western Blotting : Used For Proteins in a
sample

3. Southern Blotting
 A Southern blot is a method used
in molecular biology for detection of a
specific DNA sequence in DNA samples.
Southern blotting combines transfer
of electrophoresis-separated DNA fragments
to a filter membrane and subsequent
fragment detection by probe hybridization.
 The method is named after its inventor,
the British biologist Edwin Southern.
Other blotting methods (i.e., western
blot, northern blot, eastern
blot, southwestern blot) that employ similar
principles, but using RNA or protein, have
later been named in reference to Edwin
Southern’s name.

4. Principle of Southern Blotting
 Southern blotting is a hybridization technique for
identification of particular size of DNA from the
mixture of other similar molecules. This technique
is based on the principle of separation of DNA
fragments by gel electrophoresis and identified by
labelled probe hybridization.
 Basically, the DNA fragments are separated on
the basis of size and charge during
electrophoresis. Separated DNA fragments after
transferring on nylon membrane, the desired DNA
is detected using specific DNA probe that is
complementary to the desired DNA.
 A hybridization probe is a short (100-500bp),
single stranded DNA. The probes are labeled with
a marker so that they can be detected after

5. Procedure

6. Procedure
 Restriction digest: by RE enzyme and
amplification by PCR
 Gel electrophoresis: SDS gel
electrophoresis
 Denaturation: Treating with HCl and
NaOH
 Blotting
 Baking and Blocking with casein in BSA
 Hybridization using labelled probes
 Visualization by autoradiogram

7. Southern Blotting

8. Southern Blotting

9. Application of Southern
Blotting
 Southern blotting technique is used to detect DNA
in given sample.
 DNA finger printing is an example of southern
blotting
 Used for paternity testing, criminal identification,
victim identification
 To isolate and identify desire gene of interest.
 Used in restriction fragment length polymorphism
 To identify mutation or gene rearrangement in the
sequence of DNA
 Used in diagnosis of disease caused by genetic
defects
 Used to identify infectious agents

11. Southern Blotting
 https://www.youtube.com/watch?v=TO
KhHy7rU18

12. What is a Northern Blotting ?
 The northern blotting is a technique used in
molecular biology research to study gene
expression by detection of RNA.
 The Northern blot, also known as the RNA blot,
is one of the blotting techniques used to
transfer RNA onto a carrier for sorting and
identification.
 The Northern blot is similar to the Southern
blot except that RNA instead of DNA is the
subject of analysis in this technique.
 It is mRNA which is isolated and hybridized in
northern blots.

13. Discovery of Northern Blotting
 The northern blot technique was developed in
1977 by James Alwine, David Kemp, and George
Stark at Stanford University.
 J.C. Alwine, a biologist with a sense of humor,
developed a technique analogous to the Southern
blot, this time for the identification of a specific
RNA within a complex RNA sample using a radio-
labelled DNA probe. Alwine couldn’t resist the
temptation to call his technique the northern blot in
an allusion to Southern’s technique, raising a
chuckles in labs everywhere.

14. Principle of Northern Blotting
 Northern blotting is a method used to study gene
expression by detection of RNA in a sample.
Therefore,it is also called RNA Blot.
 The sample RNA is isolated from an organism of
interest and then electrophoresed on agarose gel
which separates the fragments on the basis of
their size.
 The separated RNA fragments are transferred to a
support membrane(nitrocellulose membrane).
This can be performed by simple capillary method
in presence of a specific buffer.

15.  The RNA is then immobilized on membrane
eitherby baking at high temperature or UV
crooslinking,which results in covalent linkage of
RNA to membrane preventing nucleic acid from
being washed away from subsequent processing.

16.  This is followed by hybridisation with a labeled
DNA or RNA probe. If the sample contains the
complementary RNA sequence, the probe will
bind to membrane to form double stranded
DNA-RNA hybrid molecule between single
stranded DNA probe and single stranded target
RNA.
 The final step is the detection of RNA of
interest on the membrane using chromogen.

17. Requirements of Northern Blotting
 Agarose Gel for process of gel electrophoresis.
 Nylon membrane/ Diazo benzyl oxy methyl (DBM)
filter paper.
 Complementary Radioactive probe for
hybridisation.
 Formaldehyde(HCHO) for degradation - Carbonal
group reacts to form stiff base with amino group of
GAC, this prevents normal H-bonding & Hence
maintain RNA in denatured State.
 X-ray film for identification of RNA.
 Note -No need of restriction enzyme for Northern
Blotting.

18. Procedure
 Step 1: DNA containing the gene of interest is
exteacted from human cells and cut into fragments
by ristriction enzymes.
 Step 2: The fragments are separated According to
size by gel electrophoresis. Each band consist of
many copies of a particular DNA fragment. Thus
bands are invisible but can be visible by straining.
 Step 3: The DNA bands are transferred to a
nitrocellulose filter by blotting. The solution passes
through the gel and filter to the paper towels.

19. Schematic view of Northern Blot
 kk

20. • Step 4: This produces a nitrocellulose filter with DNA
fragments positioned exactly as on the gel.
• Step 5: The filter is exposed to radioactivity labelled
probe for a specific gene. The Probe will base- pair
(hybridise) wiyh a short sequence present on the gene.
• Step 6: The filter is then exposed to X ray Film. The
Fragment containing the gene of interest is identified
by a band on the developed film.

22. Application of Northern
Blotting
 Observe a particular gene's
expression,pattern between tissues,organs,
development stages,environment stress
levelS etc.
 Used to show overexpression of oncogenes
and down regulation of tumour suppressor
gene's in cancerous cells.
 Detecting a specific mRNA in sample used
for screening recombinant which are
successfully transformed with transfer.
 Also used for studying mRNA Splicing.

23. Reverse Northern Blotting
 The reverse northern blot is a method by
which gene expression patterns may be
analyzed by comparing isolated RNA
molecules from a tester sample to samples
in a control cDNA library. It is a variant of the
northern blot in which the nucleic acid
immobilized on a membrane is a collection of
isolated DNA fragments rather than RNA,
and the probe is RNA extracted from a tissue
and radioactively labelled.

25. Advantages of Northern Blotting
 Northern blots are particularly useful to determine
conditions under which specific genes are
expressed.
 Only mRNA from cell types that are synthesizing
protein will hybridize to the probe.
 It is also useful in detection of mRNA transcript
size.Specifity is relatively high.
 Blots can be stored for several years and reprobed

26. Disadvantages of Northern
Blotting Risk of mRNA degradation during electrophoresis:
quality and quantification of expression are
negatively affected.
 High doses of radioactivity and formaldehyde are
a risk for workers and the environment
 Detection with multiple probes is difficult and also
time consuming procedure
 Use of ethidium bromide, DEPC and UV light
needs special training and attention.

27. Precaution used in Northern
blotting
 Remove air bubbles trapped between the gel
& the membrane.
 Ensure that all buffer components are fully
dissolved before using.
 Ensure that the electrophoresis tanks are
rinsed with distilled water after used.
 Control the temperature during hybridization.
 Always check for the incorporation of
radioactive label before using the probe.
 Don’t reuse electrophoresis buffer &
radioactive probes.

28. Western Blotting: Definition &
Principle
 Western blotting is a technique which is
used for identification of particular protein
from the mixture of protein.
 In this method labelled antibody against
particular protein is used identify the desired
protein, so it is a specific test. Western
blotting is also known as immunoblotting
because it uses antibodies to detect the
protein

29. Procedure
 Extraction of protein
 Gel electrophoresis: SDS PAGE
 Blotting: electrical or capillary blotting
 Blocking: BSA
 Treatment with primary antibody
 Treatment with secondary antibody(
enzyme labelled anti Ab)
 Treatment with specific substrate; if
enzyme is alkaline phosphatase,
substrate is p-nitro phenyl phosphate
which give color.

31. Application
 To determine the size and amount of
protein in given sample.
 Disease diagnosis: detects antibody
against virus or bacteria in serum.
 Western blotting technique is the
confirmatory test for HIV. It detects anti
HIV antibody in patient’s serum.
 Useful to detect defective proteins. For
eg Prions disease.
 Definitive test for Creutzfeldt-Jacob
disease, Lyme disease, Hepatitis B and
Herpes

33. Western Blotting
 https://www.youtube.com/watch?v=GJ
JGNOdhP8w
 https://www.youtube.com/watch?v=Jc
N0EkcHrKk
 https://www.youtube.com/watch?v=Io
VzpL_heFo

34. Precaution
 Avoid buying cheap antibodies
 Be careful when re-using antibodies
 Dissolve the milk (or blocking buffer)
properly to avoid black dots
 Use an internal standard to ease
quantification between different
membranes

36. THANK YOU