These slides include all the 3 blotting techniques i,e Southern,northern and western.Their principle,procedure and application.

1. BY:
FARHA BANU
MSC MICROBILOGY

2. SOUTHERN BLOTTING
 The technique was developed by E.M.Southern in 1975.
 The southern blot is used to detect the presence of a
particular DNA fragment in a sample.
 The DNA detected can be a single gene or it can be apart
of a larger piece of DNA such as viral genome.

3. PRINCIPLE
 The key to this method is hybridization.
 Hybridization - Process of forming a double-stranded
DNA molecule between a single-stranded DNA probe
and a single-stranded target DNA.
 There are two important features of hybridization:
 The reactions are specific-the probes will only bind to
targets with a complementary sequence.
 The probe can find one molecule of target in a mixture
of millions of related but noncomplementary
molecules.

4. General Scheme for Southern Blot

5. PROCEDURE
 1 :DNA purification
Isolate the DNA in question from the rest of the cellular
material in the nucleus.
Incubate specimen with detergent to promote cell lysis,cell
lysis frees cellular protein and DNA.
 Proteins are enzymatically degraded by incubation with
proteinase.
 DNA is purified from solution by alcohol precipitation.
 Visible DNA fibers are removed and suspended in buffer.

6.  Step 2:RESTRICTION DIGEESTION
 Cut the DNA into different sized framents using
restriction endonucleases(RE).

7.  Step 3:Gel electrophoresis
 Nucleic acid has a net negative charge and will move from
the left to right.The larger molecules are held up while the
smaller ones move faster.This results in a separation by
size.
 Gels are Agarose or polyacrylamide
With microscopic pores.
 Gel is soaked in a buffer which
Controls the size of the pores.

8.  Gels can be stained with ethidium bromide,this cause
DNA to fluoresce under UV light which permits
photography of the gel.
 This will help us to know the exact migration of DNA
strands and the quality of the RE digestion of the test
DNA.

9. Step 3. Nitrocellulose Blot
 Cover gel with nitrocellulose
paper…then…
 Cover nitrocellulose paper with
thick layer of paper towels.
 Compress apparatus with heavy
weight.
 ssDNA binds to nitrocellulose at
same position it had on the gel.
 Vacum dry nitrocellulose at 80C
to permanently fix DNA in place
or cross link (via covalent bonds)
the DNA to the membrane.

10. Step 4. Hybridization
 Incubate nitrocellulose sheet
with a minimal quantity of
solution containing 32P-labeled
ssDNA probe.
 Probe sequence is
complementary to the DNA of
interest.
 Incubate for several hours at
suitable renaturation
temperature that will permit
probe to anneal to its target
sequence(s).
 Wash & dry nitrocellulose sheet.

11. Step 5. Autoradiography
 Place nitrocellulose sheet
over X-ray film.
 X-ray film darkens where
the fragments are
complementary to the
radioactive probes.

12. General Scheme for Southern Blot

13. APPLICATIONS
 Used to diagnose sickle cell-anemia.
 Diagnosis for HIV and infectoius disease.
 To identify specific DNA in a DNA sample.
 To isolate desired DNA for construction of rDNA.
 Identify mutations,deletions,and gene
rearrangements.

14. NOTHERN BLOTTING

15. 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

16. Steps involved in Northern
Blotting
1. RNA is isolated from several biological
samples (e.g. various tissues, various
developmental stages of same tissue etc.)

17. STEPS
2.Sample’s are loaded on
gel and the RNA samples
are separated according to
their size on an agarose gel .
 The resulting gel following
after the electrophoresis
run.

18. STEPS
 The gel is then blotted
on a nylon membrane
or a nitrocellulose
filter paper by creating
the sandwich
arrangement. Nylon
membrane with a positive
charge is the most
effective for use in
northern blotting since
the negatively charged
nucleic acids have a high
affinity for them.

19. STEPS
 Once the RNA has been transferred to the
membrane, it is immobilized through covalent
linkage to the membrane by UV light or heat.

20. STEPS
The membrane is placed in a
dish containing hybridization
buffer with a labeled probe.
 Thus, it will hybridize to the
RNA on the blot that
corresponds to the sequence
of interest.
5. The membrane is washed to
remove unbound probe.

21. STEPS
 The labeled probe is detected via
autoradiography or via a
chemiluminescence reaction (if a
chemically labeled probe is used). In
both cases this results in the formation
of a dark band on an X-ray film.
 Now the expression patterns of the
sequence of interest in the different
samples can be compared.

22. NORTHERN BLOTTING

23. APPLICATIONS
 Is useful for revealing the size of the mRNA
encoded by a gene.
 Study RNA degradation
 Study RNA splicing
 Study RNA half-life
 Often used to confirm and check transgenic /
knockout mice (animals)

24. WESTERN BLOTTING

25. WESTERN BLOTTING
 Protein blotting is an analytical method that involves
the immobilization of proteins on membranes before
detection using monoclonal or polyclonalantibodies.
 There are different blotting protocols(dot blot, 2D
blot); one of the most powerful is western blotting.

26. Principle of Western Blotting
 Western blotting is an Immunoblotting technique which
rely on the specificity of binding between a molecule of
interest and a probe to allow detection of the molecule of
interest in a mixture of many other similar molecules.
 In Western blotting, the molecule of interest is a protein
and the probe is typically an antibody raised against that
particular protein.
 The SDS PAGE technique is a prerequisite for Western
blotting

27. Definition
 The Western Blot is an
analytical technique used to
detect specific proteins in a
given sample of tissue
homogenate or extract.

28. Gel electrophoresis
 Uses gel electrophoresis (SDS
PAGE) to separate native or
denatured proteins by the
length of the polypeptide
(denaturing conditions) or by
the 3-D structure of the protein
(native/ non-denaturing
conditions). The proteins are
then transferred to a membrane
(typically nitrocellulose or
PVDF), where they are probed
(detected) using antibodies
specific to the target protein.

30. The procedure includes
 Tissue preparation
 Gel electrophoresis
 Transfer
 Blocking
 Detection
 Analysis

31. Tissue Preparations
 Samples may be taken from whole tissue or from cell
culture.
 In most cases, solid tissues are first broken down
mechanically using a blender.
 It should be noted that bacteria, virus or environmental
samples can be the source of protein and thus Western
blotting is not restricted to cellular studies only.
 Assorted detergents, salts, and buffers may be employed to
encourage lysis of cells and to solubilize proteins.
 Tissue preparation is often done at cold temperatures to
avoid protein denaturing.

32. Gel Electrophoresis
 The proteins of the sample are separated using gel
electrophoresis. Separation of proteins may be by
isoelectric point, molecular weight, electric charge, or a
combination of these factors.
 The principle involved is the difference in the
ELECTROPHORETIC MOBILITIES of different proteins.

33. Transferring
 In order to make the proteins accessible to antibody
detection, they are moved from within the gel onto a
membrane made of nitrocellulose or polyvinylidene
difluoride (PVDF). The membrane is placed on top of the
gel, and a stack of filter papers placed on top of that. The
entire stack is placed in a buffer solution which moves up
the paper by capillary action, bringing the proteins with
it.

34.  Another method for transferring the proteins is called
electro blotting and uses an electric current to pull
proteins from the gel into the PVDF or nitrocellulose
membrane

35. BLOCKING
 The membrane supports used in Western blotting
have a high affinity for proteins. Therefore, after the
transfer of the proteins from the gel, it is important
to block the remaining surface of the membrane to
prevent nonspecific binding of the detection
antibodies during subsequent steps.
 A variety of blocking buffers ranging from milk or
normal serum to highly purified proteins have
been used to block free sites on a membrane. The
blocking buffer should improve the sensitivity of
the assay by reducing background interference and
improving the signal to noise ratio.

36.  Blocking of non-specific binding is achieved by placing
the membrane in a dilute solution of protein - typically
Bovine serum albumin(BSA) with a minute percentage of
detergent such as Tween 20.

37. DETECTION
 During the detection process, the membrane is "probed"
for the protein of interest with a antibody that is primary
antibody and incubated.
 Secondary antibody which is linked to a reporter enzyme
is added, which when exposed to an appropriate
substrate drives a colorimetric reaction and produces a
color.

39. ANALYSIS
 After the unbound probes are washed away, the western
blot is ready for detection of the probes that are labeled
and bound to the protein of interest.
 The membrane is then detected using the label
antibody,usually with an enzyme such as horse radish
peroxide(HRP),which is detected by the signal it
produces corresponding to the position of the target
protein.This signal is captured on a film which is usually
developed in a dark room.

41. ADVANTAGES
 While ELISA being a non specific test, Western blotting is a
more specific test for detection of HIV.
 It can detect one protein in a mixture of proteins while giving
information about the size of the protein and so is more
specific.
 Western blot is also used as the definitive test for Bovine
spongiform encephalopathy (BSE(
 Some forms of Lyme disease testing employ Western
blotting .
 It also tells you how much protein has accumulated in cells.

42. DISADVANTAGES
 If a protein is degraded quickly, Western
blotting won't detect it well.
 This test takes longer that other existing
tests .
 It might also be more costly

43. THANK YOU