BLOTTING TECHNIQUES

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BLOTTING TECHNIQUES

• M.Prasad Naidu
• MSc Medical Biochemistry, Ph.D,.

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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 .

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Types of blotting techniques

• 1 ) Southern blotting ( to detect DNA )
• 2 ) Northern blotting ( to detect RNA )
• 3 ) Western blotting ( to detect protein )

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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.

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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 ) .

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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
  .

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• 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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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 .

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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.

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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 .

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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 ß emitter .
• Bands of labeled DNA on electrophoresis gel can
  located by autoradiography .

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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.

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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 .

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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 .

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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 .

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contd

• Conditions of high stringency in hybridization
  assay are
• Low salt concentration ,
• High formamide levels ,
• 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 .

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• 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 Mg2 have stronger effect
  on hybridization .

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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 .

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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.

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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 .

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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 .

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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

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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Thank you