blotting techniques

1. BBLLOOTTTTIINNGG TTEECCHHNNIIQQUUEESS

2. DDeeffiinniittiioonn
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. TTyyppeess ooff bblloottttiinngg tteecchhnniiqquueess
1 ) Southern blotting ( to detect DNA )
2 ) Northern blotting ( to detect RNA )
3 ) Western blotting ( to detect
protein )

4. SSoouutthheerrnn BBlloottttiinngg
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. DDeeffiinniittiioonn
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. PPrroocceedduurree
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. ccoonnttdd
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. ccoonnttdd
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. ccoonnttdd
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. ccoonnttdd
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. ccoonnttdd
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. BBlloottttiinngg
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. ccoonnttdd
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. HHyybbrriiddiizzaattiioonn aassssaayyss
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. ccoonnttdd
 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. ccoonnttdd
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. AApppplliiccaattiioonnss
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. NNoorrtthheerrnn bblloottttiinngg
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. ccoonnttdd
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. AApppplliiccaattiioonnss
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. DDiissaaddvvaannttaaggeess
Time consuming procedure .
RNA samples can be degraded by RNases .
Use of radioactive probes .
Detection with multiple probes is a problem .

24. WWeesstteerrnn bblloottttiinngg
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. CCoonnttdd
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. ccoonnttdd
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. ccoonnttdd
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. AApppplliiccaattiioonnss
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. ccoonnttdd
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 .

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