The presentation aims to cover the basic principles along with a practical approach towards the Southern, Northern and Western Blot analyses.

1. Southern Blot ANALYSIS
Dr. Riddhi Datta
Department of Botany
Dr.A.P.J.Abdul Kalam Government College

2. Blotting technique
 A blot, in molecular biology and genetics, is a
method of transferring proteins, DNA or RNA or
protein, onto a carrier.
 The term "blotting" refers to the transfer of
biological samples from a gel to a membrane and
their subsequent detection on the surface of the
membrane.

3. Blotting
techniques
Southern Blot Detects DNA
Northern Blot Detects RNA
Western Blot Detects protein
Eastern Blot Detects PTM
Southwestern Blot
Detects DNA-
protein interaction
Northwestern
Blot
Detects RNA-
protein interaction

4. Southern blot
 Used to study how genes are
organized within genomes by
mapping restriction sites in and
around segments of genomic DNA
for which specific probes are
available.
 Combines transfer of electrophoresis -
separated DNA fragments to a
membrane and subsequent fragment
detection by probe hybridization.
 Named after its inventor, the British
biologist Edwin Mellor Southern.

5. Southern blot
 The technique is based on HYBRIDIZATION.
 Hybridization is the process of forming a complementary base-
pairing between a single-stranded DNA probe and a single-stranded
target DNA.
 The reactions are highly specific i.e. probes will only bind to targets
with a complementary sequence.

6. Steps of Southern blot
Step1
• Genomic DNA is first digested with one or more restriction enzymes
Step II
• Resulting fragments are separated according to size by electrophoresis through
a standard agarose gel
Step III
• DNA is then denatured in situ
Step IV
• Denatured DNA is transferred from the gel to a solid support (usually a nylon
or nitrocellulose membrane) via capillary transfer
StepV
• DNA attached to the membrane is hybridized to a labeled DNA, RNA, or
oligonucleotide probe
StepVI
• Bands complementary to the probe are located by an appropriate detection
system, for example, by autoradiography

7. Steps of Southern blot

8. Transfer methods
 The transfer of electrophoretically separated DNA from gels to two-
dimensional solid supports is a key step in Southern hybridization.
Upward CapillaryTransfer
Downward CapillaryTransfer
SimultaneousTransfer to Two Membranes
ElectrophoreticTransfer
VacuumTransfer
DNA fragments carried from gel in an
upward flow of liquid and deposited on
the surface of the solid support
DNA fragments carried in downward
flow of alkaline buffer and deposited on
the surface of a solid support
For high conc. of target DNA, capillary
method can transfer DNA
simultaneously to two solid supports
Used for analysis of small fragments of
DNA separated by PAGE. Only for
nylon membranes
DNA and RNA can be transferred
rapidly and quantitatively from gels
under vacuum

9. Upward CapillaryTransfer
 The liquid is drawn upward through the gel by capillary action.
 Rate of transfer depends on:
◦ size of the DNA fragments
◦ concentration of agarose in the gel
 Small fragments of DNA
(<1 kb) are transferred
within 1 hour
 Larger fragments are
transferred more slowly
and less efficiently.
 Capillary transfer of
DNAs > 15 kb requires
at least 18 hours

10. How we do it!
 STEP 1: Isolate genomic DNA (10 μg per lane required)
 STEP II: Restriction digestion with one or more enzymes and agarose
gel electrophoresis

11. How we do it!
 STEP III: The digested DNA fragments are denatured in presence of
alkali in situ (in gel)
 STEP IV: The fragments are then neutralized with NaCl to prevent
renaturation before addition of probe
 STEP V: Upward capillary transfer of DNA fragments to membrane
(solid support). Usually a nitrocellulose or nylon membrane is used.
 STEP VI: UV cross-linking of the transferred DNA fragments to the
membrane

12. How we do it!
 STEP VII: Preparation of radiolabelled probe usually with α-P32 labelled dCTP or
dATP. Fluorescence labelling with Digoxigenin (DIG) is also possible.
Probe Application Starting Material Method Principle
dsDNA Northern,
Southern, dot
blots
cDNA inserts, PCR
product, plasmid
with insert
Random
priming
• Use a mixture of hexa- or decanucleotides to prime
DNA synthesis randomly on single stranded DNA.
• Accomplished by Klenow fragment (lacks the 5’ →
3’ exonuclease activity)
ssRNA NPA,
in situ
hybridization
Plasmid with insert
and polymerase
promoter
In vitro
transcripti
on
• In vitro transcription requires purified linear DNA
template containing a promoter, rNTPs, a buffer system
that includes DTT and magnesium ions, and an
appropriate phage RNA polymerase (SP6 orT3 orT7)
Oligonuc
leotide
Northern,
Southern, dot
blots, NPA
Oligonucleotide End
labelling
• 3' end labeling of DNA is usually carried out using
terminal transferase in template-independent manner to
form a homopolymeric tail.
• T4 RNA ligase can be used to 3'-end label RNA
molecules.
• 5' end labeling of DNA (or RNA) is done using T4
Polynucleotide kinase with γ-P32 ATP.

13. How we do it!
 STEP VIII: Hybridization of the membrane
with labeled probe under specific
condition. The probe hybridizes with the
complementary DNA fragment.
 Before hybridization blocking is done
with salmon sperm DNA to eliminate
non-specific probe binding
 STEP IX:Wash the unbound probe.
 STEP X: Detection by autoradiography.
https://www.youtube.com/watch?v=TOKhHy7rU18

14. Application of Southern Blot
Primary usage is to identify a specific DNA
in a DNA sample.
 To confirm integration of a transgene in the
host genome.
 To identify copy number of the transgene
integrated in the host genome.

15. Application of Southern Blot
 Identify mutations, deletions, and gene rearrangements.
 In RFLP
 Used in prognosis of cancer and in prenatal diagnosis of
genetic diseases.
 Diagnosis of HIV-1 and infectious disease.
 In DNA fingerprinting:
◦ Paternity and Maternity Testing
◦ Criminal Identification and Forensics
◦ Personal Identification

16. Practical example 1
9 related genera (lanes 1-9) were screened for the presence of a particular gene
using probe for a conserved domain. Lane 10 has positive control.
For genera 1,2,5,7 and 9 the homologous sequence was absent.
For genera 2,4 and 8 the homologous sequence was present.
For genera 6 the homologous sequence was present, but with a genetic variation.

17. Practical example II
• Analysis of transgenic events using
the Southern blot technique.
• Gene was cloned using EcoRI and
BamHI.
• RE digestion for Southern blot
was done with HinDIII
C1: Positive control
C2: Negative control (wild plant)
1 to 11: Independent transgenic lines
Lines 3, 5, 8 and 10 has single copy of
transgene
Lines 2, 6 and 9 has 2 copies of transgene
Lines 1 and 11 has 3 copies of transgene
Line 7 has multiple copies of transgene
Lines 4 don’t have transgene integration.

18. Practical example III
Analysis of transgenic events using the Southern blot technique.
Gene was cloned using EcoRI and BamHI.
RE digestion for Southern blot was done with EcoRI and BamHI
‘-’ : Negative control (wild plant)
1,2,3,1c: Independent transgenic lines
5c: Positive control
The transgene has been integrated in the host genome.
Copy number of transgene can not be determined.

19. Practical example IV
• Analysis of transgenic events using the
Southern blot technique.
• Gene was cloned using XbaI and BamHI.
• RE digestion for Southern blot was done with
EcoRI (E) in one and HinDIII (H) in the other
set
AtECS1 and AtECS23 has single copy of transgene
AtECS19 have 2 copies of transgene
AtECS25 have 3 copies of transgene
Col-0 shows no integration

20. Practical examples
• Analysis of transgenic
events using the
Southern blot
technique.
• Gene was cloned using
EcoRI and BamHI.
• RE digestion for
Southern blot was done
with XbaI in one and
EcoRV in the other blot
Lanes 2,3,5,6 and 7 has single copy of transgene
Lanes 1, 9,10 and11 has 2 copies of transgene
Lanes 4 and 8 has 3 copies of transgene

21. Northern Blot Analysis

22. Northern Blot Analysis
• Northern blotting is a technique for detection of
specific RNA sequences in the transcriptome.
• Northern blotting was developed by James Alwine
and George Stark (1979) and was named such by
analogy to Southern blotting.

23. Steps of Northern blot
Step1
• Total RNA/mRNA is isolated from tissue samples
Step II
• RNA is separated according to size by electrophoresis through a standard
formaldehyde agarose gel
Step III
• RNA is then denatured in situ
Step IV
• Denatured RNA is transferred from the gel to a solid support (usually a nylon
or nitrocellulose membrane) via capillary transfer
StepV
• RNA attached to the membrane is hybridized to a labeled DNA, RNA, or
oligonucleotide probe
StepVI
• Bands complementary to the probe are located by an appropriate detection
system, for example, by autoradiography

24. Application of Northern Blot
 Used 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 transgene expression.

25. Practical examples
Detection of expression level Detection of RNA degradation

26. Western Blotting
 Western blotting is a widely used to detect specific
protein in a sample of tissue homogenate or extract.
 It works on the principle of gel electrophoresis.
 Proteins are separated based on their size on
polyacrylamide gel.

27. Steps of Western blot
I
• Total protein is isolated from tissue samples
II
• Protein is separated according to size by electrophoresis through PAGE
III
• Protein is transferred from gel to membrane by wet/semi-dry/dry electro-transfer
IV
• The membrane is stained with Ponceau to check quality of transfer and then washed to
remove the stain
V
• The membrane is blocked with skimmed milk to prevent non-specific antibody binding.
VI
• After blocking, the membrane is probed with primary antibody,followed HRP-tagged
secondary antibody.
VII
• The membrane is washed to remove unbound antibody and then substrate for HRP is added
VIII
• Bands are detected by on X-ray film or through digital imaging

28. How we do it!
 Step I: Isolate protein.
 Total protein/protein fractions
 Purified protein/crude protein
 Step II: Separate protein sample
on the basis of MW on PAGE
 Reducing/Non-reducing gel

29. How we do it!
 Step III: Transfer protein from gel to
membrane.
 PVDF (Polyvinylidene fluoride)
membrane used
 Use of a pre-stained protein ladder
helps to detect transfer process
 Electro-transfer is done. Wet/semi-
dry/dry transfer systems are
avaibable

30. How we do it!
 Step IV: Stain the membrane
with ponceau.
 Checks if the transfer is
complete
 Stain is washed with water
before stepV.
 Step V: Blocking is done with
skimmed milk to eliminate non-
specific antibody binding.

31. How we do it!
 StepVI:Antibody probing
 After blocking, the membrane is incubated
with primary antibody over night.
 This is followed by incubation with
secondary antibody (tagged with horse
radish peroxidase/alkaline phosphatase)
 The membrane is then washed to remove
unbound antibodies
 StepVII: Detection
 The membrane is treated with the substrate
for HRP/AP which gives chemiluminescence.
 The signal can be recorded on X-ray film or
in a chemi-doc.

32. Western blot detection
Colorimetric detection: HRP catalyzes a reaction with 4-Chloro-1-napthol (4CN)
and peroxide that produces a visible and insoluble purple product…outdated and low
sensitivity.
Chemiluminescence detection: In the presence of HRP and a peroxide buffer,
luminol oxidizes and forms an excited state product called 3-aminophthalate that
emits light at 450 nm….easy and high sensitivity.
Fluorescence detection: Antibodies are conjugated to a specific fluorophore and can be
detected using an imaging system…easy but moderate sensitivity.

33. Chemiluminescence detection
Alkaline phosphatase-tagged antibody
Enzymatic dephosphorylation of dioxetane substrate by alkaline
phosphatase leads to the metastable phenolate anion which, upon
decomposition, emits light at ≈480 nm.

34. HRP-tagged antibody
• Secondary antibody is
tagged with HRP
• ECL (Enhanced
chemiluminesce)
substrate contains
Luminol, hydrogen
peroxide and an
enhancer (phenol,
naphthol, etc.)
• Enhance is added so that
the reaction can proceed
for prolonged durations
• The reaction emits
light signal at 450 nm

35. Application of Western Blot
 Detection of specific protein in the proteome
 Detecting phosphorylation states of proteins using specifically
designed antibodies. Phosphorylation also makes proteins
heavier so that their position on blots get slightly shifted.
 Detecting changes in protein levels across treatment groups.
 Detection of post-translational modification of a protein (i.e.
phosphorylation, ubiquitination, etc.) using specific antibodies.