Southern, Northern, and Western blotting techniques allow for the detection of specific DNA, RNA, and protein fragments, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe for detection via hybridization. Northern blotting is similar but detects RNA. Western blotting separates proteins via gel electrophoresis, transfers them to a membrane, then uses primary and secondary antibodies for detection. These techniques are used for applications like gene mapping and the study of gene expression.

1. BLOTTINGTECHNIQUESBLOTTINGTECHNIQUES
M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2. DefinitionDefinition
Visualization of specific DNA , RNA &
protein among many thousands of
contaminating molecules requires the
convergence of number of techniques
which are collectively termed BLOT
transfer .

3. Types of blotting techniquesTypes of blotting techniques
1 ) Southern blotting ( to detect DNA )
2 ) Northern blotting ( to detect RNA )
3 ) Western blotting ( to detect
protein )

4. Southern BlottingSouthern Blotting
In 1975 Edward Southern developed this
technique that is widely used to detect
fragments of DNA .
 This requires
1 ) Separation of DNA or DNA fragments by
agarose gel electrophoresis .
2 ) DNA fragments are blotted onto a strip of
nitrocellulose or a nylon membrane.
3 ) Identification by hybridization with a
labeled ,complementary nucleic acid probe.

5. DefinitionDefinition
Southern blot a method for transferring DNA
from an agarose gel to nitrocellulose filter ,
on which the DNA can be detected by
suitable probe ( eg : complementary DNA or
RNA ) .

6. ProcedureProcedure
The DNA sample is digested by restriction
endonucleases , producing small fragments &
that are amenable for analysis .
Fragments are seperated by agarose gel
electrophoresis or PAGE .
The mobility of nucleic acids in agarose gels is
influenced by agarose concentration ,
molecular size & molecular conformation of
the nucleic acid .

7. Agarose concentration of 0.3 – 2 %are
most effective for nucleic acid
separation .
Like proteins nucleic acids migrate at rate
that is inversely proportional to the
logarithm of their molecular weight .

8. contdcontd
Separated nucleic acids are visualized by
fluorescent dye ethidium bromide .
The agarose gel is soaked in a solution of dye
& washed for remain excess dye .
illumination of the rinsed slab with UV light
reveals red orange stains where nucleic acids
are located .

9. contdcontd
Ethidium bromide stains both single & double
stranded nucleic acids , the fluorescence is
much greater with double stranded molecules
.
The electrophoresis can be performed with
dye incorporated in the gel & buffer .
This has the advantage that the gel can be
illuminated with UV light during
electrophoresis to view the extent of
separation.

10. contdcontd
The mobility of DNA may be reduced by 10
-15 % in the presence of ethidium bromide .
Ethidium bromide must be used with great
care as it is a potent mutagen .
Gloves should be worn at all times while
using the dye solutions or handling gels .

11. contdcontd
Newer fluorescent SYBR dyes produced by
molecular probes offer several advantages ,
less toxic & 5 times more sensitive than
ethidium bromide.
Labeled DNA with radioisotope P32
at 5’ & 3’
ends .
P 32
is a strong β emitter .
Bands of labeled DNA on electrophoresis gel
can located by autoradiography .

12. contdcontd
Labelling molecule before analysis with
coenzyme biotin , biotin forms a strong
complex with enzyme linked streptavidin .
PAGE is useful for analysing small fragments
of DNA upto 3,50,00 daltons ( 500 bp ) in
molecular size .
Large molecules of DNA could be separated
by pulsed field gel electrophoresis.

13. BlottingBlotting
Transfer of DNA from gel to nitrocellulose
membrane done by
1 ) Weak acid treatment to depurinate &fragment
the DNA , thus make it smaller & easier to elute
from the gel .
followed by
2) Denaturate with strong base& neutralisation
( hydrolyzes phosphodiester back bone at
depurinated sites )
single strands bind to membranes more efficiently )
A buffer is used to facilitate the transfer .

14. contdcontd
Original methods of transfer relied on
capillary action .
Vaccum or preassure systems can be used to
speed the transfer .
Faster & more efficient transfer is afforded by
the use of an electroblotter .
Electroblotting process is usually completes in
1-4 hours .

15. Hybridization assaysHybridization assays
All hybridization assays are based on the
ability of nucleic acids to form specific double
stranded hybrids .
The process requires
1 ) A probe that can target nucleic acids &
allow for specific complemenatary base
pairing .
2) A method to detect any resulting double
strands nucleic acids .

16. contdcontd
 Conditions of high stringency in
hybridization assay are
1) Low salt concentration ,
2) High formamide levels ,
3) High temparature .
As the stringency of the assay is lowered
increasing number of base mismatches are
tolerated .
conditions of high stringency require exact base
pairing .

17. The time required to hybridize the probe to a
given fraction of the target remains
proportional to the probe concentration .
The rate of hybridization reaction is
influenced by temperature & ionic strength.
Above the Tm no stable hybrids are present .
Divalent cations like Mg+2
have stronger effect
on hybridization .

18. contdcontd
Unbound probes are removed by washing
Probe bound to the membrane is detected by
autoradiogarphy , which reveals the DNA
fragments to which the probe hybridized .

19. ApplicationsApplications
Southern blots are used in gene discovery, mapping ,
evolution & development studies , diagnostics &
forensics .
Deletions / insertions .
pointmutations / polymorphisms .
Structural rearrangements .
Allow for determination of molecular weights of
restriction fragments .
Presence of particular bit of DNA in the sample.

20. Northern blottingNorthern blotting
Northern blotting is a technique for detection of
specific RNA sequences .
Developed by James alwine & George stark.
RNA molecules have defined length & much shorter
than genomic DNA it is not necessary to cleave
RNA before electrophoresis .
RNA is more susceptible to degradation than DNA .
RNA sample are separated based on size by gel
electrophoresis .

21. contdcontd
RNA is blotted on to a nylon positively
charged membrane .
The membrane is placed in a hybridization
buffer with a labeled probe ( usually DNA )
Labeled probe is detected by autoradiography
Expression patterns of sequences of interest
in different samples can be compared .

22. ApplicationsApplications
A standard for direct study of the gene
expression at the level of mRNA .
Detection of mRNA transcript size .
Study of RNA splicing – can detect
alternatively spliced transcripts .
Study RNA half life

23. DisadvantagesDisadvantages
Time consuming procedure .
RNA samples can be degraded by RNases .
Use of radioactive probes .
Detection with multiple probes is a problem .

24. Western blottingWestern blotting
Western blotting is an immunoblotting
technique which rely on the specificity of
binding between the molecule of interest & a
probe to allow detection of molecule of
interest in a mixture of many other similar
molecules .
In western blotting the molecule of interest is
a protein & the probe is typically an antibody
raised against that particular protein .

25. ContdContd
SDS PAGE technique is a prerequisite for
western blotting .
Protein sample is subjected to
electrophoresis on SDS polyacrylamide gel .
Electroblotting transfers the separated
proteins from the gel to the surface of
nitrocellulose membrane .

26. contdcontd
Blot is incubated with generic protein
( such as milk protein )which binds to any
remaining sticky places on the nitrocellulose .
An antibody which is specific for the protein
of interest ( the primary antibody Ab 1 ) is
added to the nitrocellulose sheet & reacts
with the antigen . Only the band containing
protein of interest binds the antibody forming
a layer of antibody molecules .

27. contdcontd
Following several rinses for removal of
nonspecifically bound Ab1 , the Ab1 – antigen
complex on the nitrocellulose sheet is
incubated with second antibody Ab2 , which
specifically recognizes the Fc domain of the
primary antibody & binds it . Ab 2 is
radioactive labeled or is covalently linked to
reporter enzyme which allows to visualize
protein – Ab1 – Ab2 complex .

28. ApplicationsApplications
The confirmatory HIV test employs a western
blot to detect anti HIV antibody in a human
sample .
Proteins from known HIV infected cells are
separated & blotted on a membrane then the
serum to be tested is applied in the primary
antibody incubation step.
Free antibody is washed away & a second anti
human antibody linked to an enzyme signal
can be added .

29. contdcontd
The stained bands then indicate the proteins
to which the patient serum contains
antibody .
Western blot is also used as definitive test for
bovine spongiform encephalopathy . ( mad
cow disease )
Some forms of Lyme disease testing employs
western blotting .