This document describes three types of blotting techniques - Southern blotting, Northern blotting, and Western blotting. Southern blotting is used to detect DNA fragments separated by agarose gel electrophoresis. Northern blotting detects specific RNA sequences separated by gel electrophoresis. Western blotting identifies proteins separated by SDS-PAGE gel using an antibody probe. The document provides detailed procedures and applications for each type of blotting.

1. M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

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

4. Southern 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. Definition
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. Procedure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. contd
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. contd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. contd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. contd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. contd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. Blotting
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. contd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 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. contd
 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. contd
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. Applications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 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. contd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. Applications
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. Disadvantages
Time consuming procedure .
RNA samples can be degraded by RNases .
Use of radioactive probes .
Detection with multiple probes is a problem .

24. Western 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. Contd
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. contd
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. contd
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. Applications
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. contd
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 .