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Blotting
Techniques:

What is blotting?


Blots are techniques for transferring
DNA , RNA and proteins onto a carrier so
they can be separated, and often follows
the use of a gel electrophoresis
...




Developed in 1975
...




Earned Sir Southern a
Lasker Award in 2005
...



It is a method routinely used in molecular biology for detection
of a specific DNA sequence in DNA samples
...


Cont…
...




The key to this method is Hybridization
...
The mixture of molecules is separated
...
The molecules are immobilized on a

matrix
...
The probe is added to the matrix to bind
to the molecules
...
Any unbound probes are then removed
...
The place where the probe is
connected corresponds to the location

of the immobilized target molecule
...
Digest the DNA with an
appropriate restriction
enzyme
...
The complex mixture of

fragments is subjected to gel
electrophoresis to separate
the fragments according to

size
...

BLOTTING
3
...



This procedure preserves the

distribution of the fragments in
the gel, creating a replica of the

gel on the filter
...
The

filter is incubated under

hybridization conditions with

a specific radiolabeled DNA
probe (a single DNA
fragment of short and
specific nucleotide
sequence)
...


Cont…
...
Excess probe is washed away
and the probe bound to the filter

is detected by autoradiography,
which reveals the DNA fragment
to which the probe hybridized
...


Northern Blotting:
Detection of RNA sequence:


Northern blotting is a technique for detection of specific

RNA sequences
...


Steps involved in Northern
blotting
1
...
g
...
)
* RNA is more susceptible to
degradation than DNA
...


Sample’s are loaded on
gel and the RNA samples
are separated according
to their size on an agarose
gel
...


Cont……

3
...


Cont……
4
...



Thus, it will hybridize to the RNA on the blot that
corresponds to the sequence of interest
...
The membrane is washed to remove unbound probe
...
The labeled probe is detected

via autoradiography or via a
chemiluminescence reaction (if

a chemically labeled probe is
used)
...



Now the expression patterns of
the sequence of interest in the

different samples can be
compared
...


The standard northern blot method is relatively les
sensitive than nuclease protection assays and

RT-PCR
...


Detection with multiple probes is a problem
...


If RNA samples are even slightly degraded by
RNases, the quality of the data and quantitation
of expression is quite negatively affected
...



The SDS PAGE technique is a prerequisite for

Western blotting
...


A

protein

sample

subjected

is

to

electrophoresis on an SDSpolyacrylamide gel
...


Electroblotting

transfers

the

proteins

separated

from the gel to the surface
of

a

membrane
...
The blot is incubated with a generic protein

(such as milk proteins or BSA) which binds to
any remaining sticky places on the
nitrocellulose
...
An antibody that is specific for the protein of
interest (the primary antibody - Ab1) is added
to the nitrocellulose sheet and reacts with the

antigen
...


Cont…
5
...

Ab2 is radioactively labeled, or is

covalently linked to a reporter enzyme,
which allows to visualize the proteinAb1-Ab2 complex
...


The confirmatory HIV test
...
Western blot is also used as the definitive
test for Bovine spongiform
encephalopathy (BSE(
...
Some forms of Lyme disease testing
employ Western

blotting

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