Introduction History General principle BLOTTING SOUTHERN BLOT NORTHERN BLOT WESTERN BLOT Applications Disadvantages Conclusions References

1. Blotting techniques
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Content
 Introduction
 History
 General principle
 BLOTTING
 SOUTHERN BLOT
NORTHERN BLOT
WESTERN BLOT
 Applications
 Disadvantages
 Conclusions
 References

3. Introduction
 Blotting is the technique in which nucleic acids or proteins are
immobilized onto a solid support generally nylon or nitrocellulose
membranes.
 Blotting of nucleic acid is the central technique for hybridization
studies.
 Blotting techniques are used to identify unique proteins and nucleic
acid sequences.

4. History
 Southern Blotting is named after its inventor,
the British biologist Edwin Southern (1975)
 Other blotting methods (i.e. western blot, WB,
northern blot, NB) that employ similar
principles, but using protein or RNA, have
later been named in reference to Edwin
Southern's name.

5. General principle
 The blotting methods are fairly simple and usually consist of four
separate steps: electrophoretic separation of protein or of nucleic acid
fragments in the sample; transfer to and immobilization on paper
support; binding of analytical probe to target molecule on paper; and
visualization of bound probe.
 Molecules in a sample are first separated by electrophoresis and then
transferred on to an easily handled support medium or membrane.
 This immobilizes the protein or DNA fragments, 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.

6. BLOTTING
 SOUTHERN BLOT
 NORTHERN BLOT
 WESTERN BLOT

7. 1. SOUTHERN BLOT
 A Southern blot is a method used 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.
 The process of Southern Blotting was created in the 1970’s by
Edward M. Southern, a scientist at Edinburgh University in
Edinburgh, Scotland. Southern blotting is designed to locate a
particular sequence of DNA within a large, complex sample of DNA.
For example, Southern Blotting can locate a single specific gene
within an entire genome (Khalsahttp, 2000).

8. Steps
 Digestion of genomic DNA (w/ ≥ one RE)  DNA fragments
 Size-separation of the fragments (standard agarose gel
electrophoresis)
 In situ denaturation of the DNA fragments (by incubation @ ↑temp)
 Transfer of denatured DNA fragments into a solid support (nylon or
nitrocellulose).
 Hybridization of the immobilized DNA to a labeled probe (DNA,
RNA)
 Detection of the bands complementary to the probe (e.g. by
autoradiography)

11. Applications
 Southern hybridization can also be used to locate the exact position
of a cloned gene within a recombinant DNA molecule.
 This is important as often the cloned DNA fragment is relatively
large (40 kb for a cosmid vector) whereas the gene of interest,
contained somewhere in the cloned fragment, may be less than 1kb
in size. Southern blots of cloned genomic DNA fragments can be
probed with cDNA molecules to find which parts of the genomic
clone correspond to the cDNA fragment.
 If the Southern blot contains genomic DNA fragments from the
whole genome, the probe will give information about the size of the
fragment the gene is on the genome and how many copies of the
gene are present in the genome.

12. Northern Blotting
 A northern blot is a method routinely used in molecular biology for
detection of a specific RNA sequence in RNA samples.
 The method was first described in the seventies (Alwine et al. 1977,
1979)
 It is still being improved (Kroczek 1993), with the basic steps
remaining the same

13. Basis Steps of NB
1. Isolation of intact mRNA
2. Separation of RNA according to size (through a denaturing
agarose gel e.g. with Glyoxal/formamide)
Transfer of the RNA to a solid support
Fixation of the RNA to the solid matrix
Hybridization of the immobilized RNA to probes
complementary to the sequences of interest
Removal of probe molecules that are nonspecifically bound to
the solid matrix
Detection, capture, & analysis of an image of the specifically
bound probe molecules.

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

16. Advantages of Northern Blotting
 It is a widely accepted and well regarded method.
Northern blotting is a straight-forward method.
 Often it is used as a confirmation or check.
 It is a versatile protocol as it can allow the usage
of many types of probes including: radiolabel and
non- radiolabeled, in vitro transcribed RNA and
even oligonucleotides such as primers.

17. Disadvantages of Northern Blotting
 Often radioactivity is used. This prevents ease of performing it,
use and disposal. New methods of non-radioactive detection
have been generated allowing non-radioactive detection.
 The whole process of northern blotting takes a long time
usually, from sample preparation to detection.
 If RNA samples are even slightly degraded by RNases, the
quality of the data and quantisation of expression is quite
negatively affected.

18. Western Blotting
“Immunoblotting”
 Electrophoretic transfer of proteins from gels to membranes.
 The western blot (alternatively, immunoblot) is used to detect
specific proteins in a given sample of tissue homogenate or
extract.
 The method originated from the laboratory of George Stark at
Stanford. The name western blot was given to the technique by
W. Neal Burnette.

19. What is Western Blotting?
 A technique in which proteins are separated
by gel electrophoresis and transferred to a
membrane sheet. A specific protein is then
identified through its reaction with a labeled
antibody.

20. Steps in a Western Blot
 The first step is gel electrophoresis.
(The proteins of the sample are separated according to
size on a gel.)
 The second is Membrane Transfer.
(The proteins in the gel are then transferred onto a
membrane made of nitrocellulose by applying current.)
 The third step is Blocking.
(Blocking is used to prevent non-specific protein
interactions between the membrane and the antibody
protein.)

21. Incubation
 Incubation occurs by diluting an antibody into a
solution which will keep the pH neutral. The majority
of the time the solution is non fat dry milk.
 The Primary antibody is then incubated into the
membrane.
 The primary antibody recognizes only the protein of
interest, and will not bind any of the other proteins
on the membrane.

22. Incubation Continued
 After rinsing the membrane to remove unbound primary antibody a
secondary antibody is incubated with the membrane.
 It binds to the first antibody.
 This secondary antibody is usually linked to an enzyme that can
allow for visual identification of where on the membrane it has
bound.
 Finally, the reaction product may produce enough fluorescence to
be detected on a sensitive sheet of film when it is placed against the
membrane.

24. Applications
 Western blotting can be used to identify a
specific antibody in a mixture.
 A western blot is also used as the definitive
test for mad cow disease.
 Western blot can also be used as a
confirmatory test for Hepatitis B infection.

25. Eastern blotting
 It is a technique to detect protein post translational modification and
is an extension of the biochemical technique of western blotting.
Proteins blotted from two dimensional SDS-PAGE gel on to a
nitrocellulose membrane are analyzed for post-translational protein
modifications using probes specifically designed to detect lipids,
carbohydrate, or any other protein modification.
 The technique was conceptualized by S. Thomas while working on
sandal spike phytoplasma and developed at the Dept. of Pathology,
University of Texas Medical Branch, Galveston, Texas, while
working on the intracellular bacteria, Ehrlichia.

26. Applications of Blotting and Hybridization Techniques
 Southern blotting technique is widely used to find specific
nucleic acid sequence present in different plant species.
 Northern blotting technique is widely used to find gene
expression and regulation of specific genes.
 By using blotting technique we can identify infectious agents
present in the sample.
 We can identify inherited disease.
 It can be applied to mapping restriction sites in single copy
gene.

27. Disadvantages of Blotting and Hybridization
Techniques
 The process is a complex, cumbersome and time consuming one.
 It requires electrophoretic separation.
 Only one gene or RNA can be analysed at a time.
 Gives information about presence of DNA, RNA or proteins but does
not give information about regulation and gene interaction.

28. Conclusions
 Identifying and measuring specific proteins in complex biological
mixtures, such as blood, have long been important goals in scientific and
diagnostic practice.
 More recently the identification of abnormal genes in genomic DNA has
become increasingly important in clinical research and genetic
counseling.
 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.

29. References
 Brown, T.A. (1998). Gene cloning an introduction, 3rd Edn. Stanley
Thornes (Publishers) Ltd.
 Kuby, Janis etal.(2003). Immunology, 5th Edn. W.H. Freeman &
Company Publishers.
 www.wikipedia.org