Blotting techniques

BLOTTING TECHNIQUES
DIPESH TAMRAKAR
M.Sc. Clinical Biochemistry
1

Definition
Visualization of specific DNA, RNA & protein by BLOT transfer
Types:
1. Southern
blotting
( to detect
DNA )
1.2. Northern
blotting
2.( to detect
RNA )
1.3. Western
blotting
2.( to detect
protein )
1.4. Eastern
blotting
2.( to detect
protein of post
translational
modifications )
2

1. Southern Blotting
The technique was developed by E.M. Southern in 1975
Used to detect the presence of a particular fragment of DNA in a
sample
This DNA can be:
• Single gene
• Part of a larger piece of DNA s/a viral genome
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 )
3

“Used to detect the DNA”
• This method Involves:
1. Separation
2. Transfer
3. Hybridization.
“The key to this method is Hybridization”
4

DNA
Each individuals unique genetic blueprint is stored in material known
as DNA.
DNA is found in all cells containing a nucleus.
DNA can be extracted for analysis from hair, bones, saliva, sperm, skin,
organs, all body tissues and blood.
Temperature Storage for DNA
Purified DNA may be refrigerated at 4°C for up to 3 years.
Samples kept over 3 years should be frozen at -70°C.
• Specimens used in DNA testing:
• Whole blood, Solid tissue, Serum and plasma
• Urine, Bone marrow and many others
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1. Separation
The DNA sample is digested by restriction endonucleases , producing
small fragments & that are amenable for analysis .
Fragments are separated 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 .
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 .
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Separated nucleic acids are visualized by fluorescent dye ethidium
bromide
The agarose gel is soaked in a solution of dye & washed for remaining
excess dye
illumination of the rinsed slab with UV light reveals red orange stains
where nucleic acids are located
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.
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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
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
P32 is a strong β emitter
Bands of labeled DNA on electrophoresis gel can located by
autoradiography
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• Labelling molecule before analysis with coenzyme biotin , biotin forms
a strong complex with enzyme linked streptavidin .
• PAGE is useful for analyzing small fragments of DNA upto 500 bp
• Large molecules of DNA could be separated by pulsed field gel
electrophoresis.
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• Ethidium bromide is an intercalating agent commonly used as a
fluorescent tag
• It is commonly abbreviated as "EtBr", which is also an abbreviation for
bromoethane
• When exposed to ultraviolet light (210 nm), it will fluoresce with an
orange colour, intensifying almost 20-fold after binding to DNA
• EtBr emits orange light with wavelength 605 nm.
Ethidium bromide
intercalated
between two
adenine-thymine
base pairs.
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2. 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 & neutralization ( hydrolyzes
phosphodiester back bone at depurinated sites )
• single strands bind to membranes more efficiently
• A buffer is used to facilitate the transfer .
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a. Capillary blotting:
The gel is placed on a wet pad of buffer-
soaked filter paper & sheet of nitrocellulose
placed on the gel
Buffer is then drawn through the gel by
placing a pad of dry absorbent material
(usually filter paper) followed by a heavy
weight on top of the sheet
Passage of buffer by capillary action through
the gel carries the separated nucleic acids on
the nitrocellulose sheet to which they bind
irreversibly by hydrophobic interaction
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b. Electro blotting:
Quicker & more efficient method of transfer
Sandwich of gel & nitrocellulose compressed in
a cassette & immersed in buffer between two
parallel electrodes
A current is passed at right angles to the gel
which causes the separated nucleic acids to
electrophorese out of the gel & into
nitrocellulose sheet
Obtained blot can be analyzed
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3. Hybridization
The key to this method is hybridization.
Hybridization-process of forming a double-stranded DNA molecule
between a single-stranded DNA probe and a single-stranded target
patient DNA.
There are 2 important features of hybridization:
The reactions are specific-the probes will only bind to targets with
a complementary sequence.
The probe can find one molecule of target in a mixture of millions
of related but non-complementary molecules.
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• Steps for hybridization:
1. The mixture of molecules are separated.
2. The molecules are immobilized on a matrix.
3. The probe is added to the matrix to bind to the molecules.
4. Any unbound probes are then removed.
5. The place where the probe is connected corresponds to the
location of the immobilized target molecule detected by
autoradiogarphy
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Probe labelling & detection:
1. Radioactively labeled, often with 32P because of its high energy and
ease of incorporation into the phosphate groups of dNTPs &
detected using X-ray film.
2. Colorimetric detection (colored complex);
a. Alkaline Phosphatase that acts on substrates 5-bromo-4-chloro-
indoylphosphate (BCIP) to produce a dark purple product)
b. Horse Reddish Peroxidase (HRP) acts on H2O2 to produce insoluble brown
product
c. HRP acts on 4-chloro-1-napthol into insoluble blue product
3. Fluorescent detection
4. Chemiluminescence detection (luminol, isoluminol)
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Applications
Southern blots are used in gene discovery, mapping , evolution &
development studies , diagnostics & forensics .
Allow for determination of molecular weights of restriction fragments
Presence of particular bit of DNA in the sample.
Identify: mutations, deletions, insertions and gene rearrangements
Used in prognosis of cancer and in prenatal diagnosis of genetic diseases
Leukemias
Diagnosis of HIV-1 and infectious disease
Applications of DNA fingerprinting include:
• Paternity and Maternity Testing
• Criminal Identification and Forensics
• Personal Identification
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Steps in southern blotting
The DNA is digested
Fragments
Gel electrophoresis
Transfer to membrane
Probing
Autoradiogram
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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
• Northern blotting was developed
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• 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 .
Northern blotting
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Applications
• A standard for the direct study of gene expression at the level of mRNA
(mRNA transcripts)
• Detection of mRNA transcript size
• Study RNA degradation
• Study RNA splicing - can detect alternatively spliced transcripts
• Study RNA half-life
• Study IRES (Internal Ribosomal Entry Site) – to remove possibility of
RNA digestion vs. 2nd cistron translation.
Northern blotting
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Disadvantages
• Time consuming procedure
• RNA samples can be degraded by RNAses
• Use of radioactive probes
• Detection with multiple probes is a problem
Northern blotting
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Steps involved in Northern Blotting
RNA isolation
Loading of sample on Agarose gel
Blotting on nitrocellulose membrane
Labeling with probe
Washing to remove unbound probe
Detection by autoradiogram
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Western blotting
• Dr. Douglas Lake of the University of Arizona School of Medicine's
Department of Microbiology and Immunology
• 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 .
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• 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
• Blot is incubated with primary and secondary antibodies on the
nitrocellulose
• Detection by autoradiography or fluorescence or colorimetric or
chemiluminescence detection
Western blotting
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Blocking
• Used to prevent the interactions between the membrane and the
antibody used for detection of the target protein.
• Blocking of non-specific binding is achieved by placing the membrane in a
dilute solution of protein – typically 3–5% bovine serum albumin (BSA) or
non-fat dry milk in tris-buffered saline (TBS), with a minute percentage
(0.1%) of detergent such as Tween 20 or Triton X-100.
• The protein in the dilute solution attaches to the membrane in all places
where the target proteins have not attached.
• Thus, when the antibody is added, there is no room on the membrane for
it to attach other than on the binding sites of the specific target protein.
• This reduces background in the final product of the western blot, leading
to clearer results, and eliminates false positives.
Western blotting
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Tissue preparation
• Samples can be taken from whole tissue or from cell culture.
• Solid tissues are first broken down mechanically using a blender using a
homogenizer or by sonication.
• Assorted detergents, salts, and buffers may be employed to encourage
lysis of cells and to solubilize proteins.
• Protease and phosphatase inhibitors are often added to prevent the
digestion of the sample by its own enzymes.
• A combination of biochemical and mechanical techniques – comprising
various types of filtration and centrifugation – can be used to separate
different cell compartments and organelles.
Western blotting
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• 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
• 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 .
Western blotting
30

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 .
Western blotting
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• 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
• Confirmatory test for Hepatitis B infection and HSV-2 infection
Western blotting
32

Western blotting in Biochemistry
study different properties of protein based on molecular weight
Helps in confirm effective inhibition of myocardial mTOR kinase
activities by analyzing protein molecular wt. Vs expected values
investigations of mitochondrial uniporter selective calcium channels
in the organelle's inner membrane: Using the western blot technique,
researchers were able to determine through phylogenetic analysis
that the uniporter must have been a feature of the earliest
mitochondria.
Also used in Blood Doping to detect drug abuse in sports
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Eastern Blotting
• developed by Towbin in 1979
• The eastern blot is a biochemical technique used to analyze protein
post translational modifications (PTM) such as lipids, phosphomoieties
and glycoconjugates.
• It is most often used to detect carbohydrate epitopes.
• Thus, eastern blotting can be considered an extension of the
biochemical technique of western blotting.
• Multiple techniques have been described by the term eastern blotting,
most use proteins blotted from SDS-PAGE gel on to a nitrocellulose
membrane.
35

• Transferred proteins are analyzed for post-translational modifications
using probes that may detect lipids, carbohydrate, phosphorylation or
any other protein modification.
• Eastern blotting should be used to refer to methods that detect their
targets through specific interaction of the PTM and the probe,
distinguishing them from a standard far-western blot.
• In principle, eastern blotting is similar to lectin blotting (i.e. detection
of carbohydrate epitopes on proteins or lipids).
Eastern Blotting
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Application
• detection of protein modifications in bacterial species Ehrlichia- E.
muris and IOE.
• The technique showed that the antigenic proteins of the non-virulent
E. muris is more post-translationally modified than the highly virulent
IOE
• PMT play an important role in translocation across biological
membranes.
• Expression of post-translated proteins is important in several diseases
Eastern Blotting
37

• Far-eastern blotting is a technique developed in 1994 by Taki and
colleagues at the Tokyo Medical and Dental University, Japan for the
analysis of lipids separated by high-performance thin layer
chromatography (HPTLC).
• The lipids are transferred from the HPTLC plate to a polyvinyledene
difluoride (PVDF) membrane for further analysis, for example by
enzymatic or ligand binding assays or mass spectrometry.
Far-Eastern blotting
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Far-Western blotting
• Far-western blotting is a molecular biological method which is based
on the technique of western blotting to detect protein-protein
interaction in vitro.
• While usual western blotting uses an antibody to detect a protein of
interest, far-western blotting uses a non-antibody protein, which can
bind the protein of interest.
• Thus, whereas western blotting is used for the detection of certain
proteins, far-western blotting is rather employed to detect
protein:protein interactions.
39

Northwestern blot
• The Northwestern blot, also known as the Northwestern assay, is a
hybrid analytical technique of the Western blot and the Northern
blot,
• used in molecular biology to detect interactions between RNA and
proteins.
• The Northwestern blot combines the two techniques, and specifically
involves the identification of labeled RNA that interact with proteins
that are immobilized on a similar nitrocellulose membrane.
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