BLOTTING TECHNIQUES

 Blotting is a powerful and sensitive technique for identifying

INTRODUCTION
 Subsequently the method was modified to detect other
targets
 The nomenclature of these techniques was built around Dr
Southern’s name resulting in the terms
 results in transfer of DNA molecules, usually restriction
fragments ,from an electrophoresis gel to a nitrocellulose or
nylon sheet in such a way that the DNA banding pattern
present in the gel is reproduced on the membrane.
 During transfer or as a result of subsequent treatment ,the
DNA becomes immobilized on the membrane and can be
used as a substrate for hybridization analysis with labelled
DNA or RNA probes that specifically target individual restric-
tion fragments in the blotted DNA.
 Southern blotting is therefore a method for ‘detection of a
specific restriction fragment against a background of many
other restriction fragments’

SOUTHERN BLOTTING
 Preparation of genomic DNA
 Cells containing the DNA to be analysed must be broken up
under relatively gentle conditions.
 For tissue culture and blood samples,SDS is preferred and for
bacteria surrounded by cellwall,EDTA or lysosyme is used.
 After cell disruption,the major biochemicals other than the
DNA present in the initial extract is removed
 The precipitated DNA is then dried and is digested with
restriction enzyme

METHODOLOGY
 DNA BLOTTING TECHNIQUES
 After restriction digestion, the complex mixture of fragments
is subjected to gel electrophoresis to separate the fragments
according to size.
 The gel has to be pre
treated before setting up
the southern blot.
 It has 2 objectives
 Break the DNA molecule in
individual bands within the gel
into smaller fragments as it
Transfer quickly than larger ones
 This is done by soaking the gel in HCL for 30 mins
 Second pre treatment is with an alkaline solution that denature the
double stranded DNA into single strand
 This aids their transfer and subsequent binding to the membrane
 Followed by neutralisation of gel by soaking in a tris salt buffer if
nitrocellulose membrane is used
 The southern blot is then setup with high salt transfer buffer
comprising Nacl and sodium citrate
 After electrophoresis, the agarose gel containing the
fractionated restriction fragments is placed on a filter paper
wick that forms a connection between the gel and a reservoir
of high salt buffer
 The nitrocellulose membrane is placed on the top of the gel
and covered with a tower of paper towels that are held in
place with a weight
 Capillary action results in the
buffer soaking through the filter
paper wick, gel and membrane
and Into paper towels.
 As the buffer passes through the gel the DNA fragments are
carried with it Into the membrane where they become bound
to nitrocellulose
 The membrane is then baked in a vaccum or regular oven at
80C for 2 hrs or exposed to UV radiation to permanently
attach the transferred DNA to the membrane
 Hybridization analysis is based on the principle that two
polynucleotides will form a stable hybrid by base-pairing if
their nucleotide sequences are wholly or partly
complementary.
 A specific restriction fragment in a Southern blot can
therefore be detected if the membrane is probed with a
second ,labelled DNA molecule that has the same ,or similar
,sequence as the fragment being sought
 Hybridization analysis is carried out by soaking the
Southern blot in a buffer containing the hybridization probe
,usually in a tube that is constantly rotated so all parts of the
membrane are exposed to the probe ,or alternatively in a
sealed plastic bag that is placed on a shaker.

DNA/DNA HYBRIDISATION
 Hybridization is carried out in two stages. First ,the
membrane is prehybridized in a solution designed to block
the unused DNA binding sites on the membrane surface. If
this step is omitted then the probe will bind non specifically
to the surface of the membrane and the signal resulting from
hybridization to the specific restriction fragment will be
difficult if not impossible to identify.
 The second stage is the actual hybridization ,which is carried
out in a high-salt buffer containing a detergent,
 Washing steps carried out after hybridization are designed
simply to remove the non hybridized probe.
 After washing ,the membrane is subjected to the detection
procedure appropriate for the label that has been used ,for
example autoradiography for a radioactive or fluorescent
probe or the development of colour on the membrane if a
chromogenic detection method is used
 Hybridisation of the probe to a specific DNA fragment on the
filter membrane indicates that this fragment contain DNA
sequence that is complementary to the probe
 Identification and cloning of a specified gene
 Southern blotting of genomic DNA is used to identify one ore
more restriction fragments that contain the gene being
sought
 Southern blotting of restricted clone DNA is used to confirm
the clone identification and to locate a shorter restriction
fragment containing the sequence of interest from within the
cloned DNA
 Major application is the technique called restriction fragment
length polymorphism mapping which is important for
construction of genome maps

APPLICATIONS
 Immuno blotting technique which rely on the specificity of
binding between molecule of interest and a probe to allow
the detection of the molecule of interest in a mixture of many
other molecule
 This method originated from the laboratory of George stark
at Stanford
 The molecule of interest is a protein and probe is an antibody
raised against that particular protein

WESTERN BLOTTING
 Tissue preparation
sample may be taken from whole tissue or from cell
culture.
 Solid tissues are broken down mechanically using a blender
or homogeniser
 Assorted detergents, salts and buffers may be employed to
encourage lysis of cells and to solubilise proteins
 Tissue preparation done at cold temperature to avoid protein
denaturing

METHODOLOGY
 Electrophoresis
SDS PAGE is a pre-requisite for western blotting
 It maintains polypeptide in a denatured state once they have
been treated with strong reducing agents to remove
secondary and tertiary structure and allows separation of
protein by molecular weight
 Sampled protein become covered with negatively charged
SDS and move to positively charged electrode through
acrylamide mesh of the gel
 Transfer
to make the protein accessible to antibody detection,
they are moved from within the gel onto a nitrocellulose
membrane
 A sandwich of gel and nitrocellulose is compressed in
cassette and immersed in buffer between two parallel
electrodes
 A current is passed at right angles to the gel which causes the
separated proteins to electrophorese out of the gel and into
nitrocellulose sheets.
 The nitrocellulose with its transferred protein is referred to
as a blot
 Probe blotting
once transferred into nitrocellulose the separated
proteins can be examined further
 this involves probing the blot usually in an antibody to detect
a specific protein
 The blot is first incubated in a protein solution which will
block all the remaining hydrophobic binding sites in the
nitrocellulose sheet
 The blot is then incubated in a dilution of an
antiserum(primary antibody)directed against the protein of
interest
 This IgG molecule will bind to its antigen thus identifying the
protein of interest
 Detection methods
 Inorder to visualise this interaction,the blot incubated further
in a solution of a secondary antibody which is directed
against the IgG of the species that provided the primary
antibody
 Most common detection method is to use an enzyme linked
secondary antibody.
 The blot is incubated in enzyme substrate solution,when the
enzyme converts the substrate into a insoluble coloured
product that is precipitated on to the nitrocellulose
 The presence of coloured band indicates the position of
protein of interest
 Radioactivity labelled DNA can be used to detect DNA binding
protein on a blot.
 The blot is first incubated in a solution of radiolabelled
DNA,then washed and autoradiograph of the blot made.
 The presence of radioactive bands detected on the
autoradiograph identifies the position of DNA binding
proteins on the blot
 Chemiluminescent detection method depends on incubation
of the western blot with a substrate that will luminescent
when exposed to the reporter on the secondary antibody
 The light is then detected by photographic film and more
recently by CCD cameras which captures a digital image of
the western blot
 The image is analysed by densitometry which evaluates the
relative amount of protein staining and quantifies the result
in terms of optical density
 Detect the presence of biological probes(circulating
antibodies)to a single protein or group of proteins
 Western blot is also used as the definitive test for
bovine spongiform encephalopathy
 Some forms of lyme disease employ western
blotting

APPLICATIONS
 It is a technique used to study the gene expession by
detection of RNA in a sample.
 Within northern blot,it is possible to observe cellular control
over structure and function by determining the particular
gene expression level during differentition,morphogenesis as
well as abnormal or diseased condition
 The technique was developed in 1977 by James alwine,David
kemp and George stark at the Stanford university
 The main difference is that RNA rather than DNA is analysed
in the northern blot.

NORTHERN BLOTTING
 Blotting technique starts with the extraction of total RNA
from a homogenised tissue sample
 RNA samples are then separated by gel electrophoresis
 A nylon membrane with positive charge is most effective
since negatively charged nucleic acids have a high affinity for
them.
 The transfer buffer used usually contains formamide because
it lowers the annealing temperature of the probe-RNA
interaction preventing RNA degradation at high temperature
 Once the RNA has been transferred to the membrane it is
immobilised through covalent linkage to the membrane by
UV light or heat

METHODOLOGY
 After a probe has been labelled it is hybridised to the
RNA on the membrane
 The membrane is washed to ensure that the probe
has bound specifically
 The hybrid signals are then detected by X ray film
and can be quantified by densitometry
 Allows in observing a particular genes expression
pattern between tissues,organs,developmental
stages environmental stress levels and pathogen
infection
 Technique widely used to show over expression of
oncogenes and down regulation of tumor
suppressor genes in cancerous cells when compared
to normal tissues as well as the gene regulation in
the rejection of transplanted organs

APPLICATIONS