BLOTTING

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BLOTTING

• Dr. Azhar Chishti
• Department of Medical Biochemistry

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LECTURE OUTLINES

1. Southern Blotting
2. History
3. Main use
4. Advantages
5. Probes
6. Hybridization
7. Procedure
8. Steps
9. Methods of Transfer
10. Example of application of SB for the diagnosis of
    diseases (SCA)
11. Northern Blotting
12. History
13. Definition
14. Basic steps
15. Applications

• Western Blotting
• WB Definition
• Applications Advantages
• WB An overview
• Direction of transfer
• Factors Affecting Transfer Efficiency
• WB procedure, briefly
• WB Detection methods
• Examples of used substrates
• WB procedure, illustrated
• Comparison between SB WB (Similarities
  Differences)

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OBJECTIVES

• To understand the basic concept of blotting
  techniques (Southern, northern, western)
• To know the main applications and advantages of
  each of the main types of blotting techniques
• To be familiar with the steps (in brief) for
  performing a blotting procedure
• To understand the major similarities
  differences between different blotting techniques
• To be introduced to an example of applying a
  blotting technique in diagnosis of diseases (SCA)

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BLOTTING
1. SOUTHERN BLOT 2. NORTHERN BLOT 3.
WESTERN BLOT
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Blotting History

• Southern Blotting is named after its inventor,
  the British biologist Edwin Southern (1975)
• Other blotting methods (i.e. western blot, WB,
  northern blot, NB) that employ similar
  principles, but using protein or RNA, have later
  been named in reference to Edwin Southern's name.

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SOUTHERN BLOTTING ?

• Experimental procedure
• DNA is extracted from cells, leukocytes.
• DNA is cleaved into many fragments by
  restriction enzyme (BamH1, EcoR1 etc)

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• The resulting fragments are separated on the
  basis of size by electrophoresis.
• The large fragments move more slowly than the
  smaller fragments.
• The lengths of the fragments are compared with
  band of relative standard fragments of known
  size.

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• The DNA fragments are denatured and transferred
  to nitrocellulose membrane (NYTRAN) for analysis.
  
• DNA represents the individual's entire genome,
  the enzymic digest contains a million or more
  fragments.
• The gene of interest is on only one of these
  pieces of DNA.

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• DNA segments were visualized by a nonspecific
  technique, they would appear as an unresolved
  blur of overlapping bands.
• To avoid this, the last step in Southern blotting
  uses a probe to identify the DNA fragments of
  interest.

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• Southern blot analysis depend on the specific
  restriction endonuclease
• The probe used to visualize the restriction
  fragments.

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Probes

• Labeled material to detect a target.
• For DNA 20-30 nucleotides, complementary to a
  region in the gene
• Methods of labeling

• Radioactive e.g. 32P

• Non-radioactive e.g. Biotin

• Sensitive
• Relatively cheap
• Hazardous
• You should follow the radioactive waste disposal
  regulations.

• Sensitive
• Relatively expensive

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Hybridization
The binding between ss labeled probe to a
complementary nucleotide sequence on the target
DNA. Degree of hybridization depends on method
of probe labeling (radioacitve or non-radioactive
system e.g. biotin-avidin.
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• Detection of mutations
• The presence of a mutation affecting a
  restriction site causes the pattern of bands to
  differ from those seen with a normal gene.
• A change in one nucleotide may alter the
  nucleotide sequence so that the restriction
  endonuclease fails to recognize and cleave at
  that site
• (for example, in Figure, person 2 lacks a
  restriction site present in person 1).

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1- DNA extraction
6- Detection
2- DNA cleavage (RE)
5- Hybridization e.g. with 32P-labeled probe
3- DNA Electrophoresis (based on size)
-

4- DNA Denature, Transfer, blocking,
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Steps

• Digestion of genomic DNA (w/ one RE) ? DNA
  fragments
• Size-separation of the fragments (standard
  agarose gel electrophoresis)
• In situ denaturation of the DNA fragments (by
  incubation _at_ ?temp)
• Transfer of denatured DNA fragments into a solid
  support (nylon or nitrocellulose).
• Hybridization of the immobilized DNA to a labeled
  probe (DNA, RNA)
• Detection of the bands complementary to the probe
  (e.g. by autoradiography)
• Estimation of the size number of the bands
  generated after digestion of the genomic DNA w/
  different RE ? placing the target DNA within a
  context of restriction sites)

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METHODS OF TRANSFER
Upward Capillary Transfer
Downward Capillary Transfer
Simultaneous Transfer to Two Membranes
Electrophoretic Transfer
Vacuum Transfer
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Example of TransferUpward Capillary Transfer
Weight
Glass Plate
Paper towels
Whatman 3MM paper
Membrane (nylon or nitrocellulose)
Whatman 3MM paper
Gel
Transfer buffer
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weight ? tight connection
DNA eluted from the gel by the moving stream of
buffer is deposited onto a membrane
Buffer drawn from a reservoir passes through the
gel into a stack of paper towels
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Example of Application of SB in diagnosis of
mutation in ? globin gene
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Example of Application of SB in diagnosis of
mutation in ? globin gene
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Northern BlottingNorthern Hybridization
A northern blot is a method routinely used in
molecular biology for detection of a specific RNA
sequence in RNA samples.

• The method was first described in the seventies
  (Alwine et al. 1977, 1979)
• It is still being improved (Kroczek 1993), with
  the basic steps remaining the same

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Basis Steps of NB

• Isolation of intact mRNA
• Separation of RNA according to size (through a
  denaturing agarose gel e.g. with
  Glyoxal/formamide)
• Transfer of the RNA to a solid support
• Fixation of the RNA to the solid matrix
• Hybridization of the immobilized RNA to probes
  complementary to the sequences of interest
• Removal of probe molecules that are
  nonspecifically bound to the solid matrix
• Detection, capture, analysis of an image of the
  specifically bound probe molecules.

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Applications

• Study of gene expression in eukaryotic cells
• To measure the amount size of RNAs transcribed
  from eukaryotic genes
• To estimate the abundance of RNAs
• Therefore, it is crucially important to equalize
  the amounts of RNA loaded into lanes of gels

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Examples of methods to equalize the amounts of
RNA loaded into lanes of gels

• OD260
• Use of housekeeping gene (endogenous
  constitutively-expressed gene) Normalizing
  samples according to their content of mRNAs of
  this housekeeping gene

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Western Blotting Immunoblotting
electrophoretic transfer of proteins from gels
to membranes
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WB Definition

• Blotting is the transfer of separated proteins
  from the gel matrix into a membrane, e.g.,
  nitrocellulose membrane, using electro- or
  vacuum-based transfer techniques.

Towbin H, et al (1979). "Electrophoretic transfer
of proteins from polyacrylamide gels to
nitrocellulose sheets procedure and some
applications.". Proc Natl Acad Sci U S A. 76 (9)
43504354
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Applications Advantages
Applications To determine the molecular weight
of a protein (identification) To measure
relative amounts (quantitation) of the protein
present in complex mixtures of proteins that are
not radiolabeled (unlike immunoprecipitation) Adva
ntages WB is highly sensitive technique As
little as 1-5 ng of an average-sized protein can
be detected by WB
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• Western blotting
• The main steps of blotting technique in a
  chronological order will be as follows
• Blocking
• Probing with the specific antibody(ies)
• Wash
• Detection
• Washing
• X-ray (Gel Documentation System)

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Electrophoretic Transfer An Overview
Important Issue Where to put the gel and the
membrane relative to the electroblotting transfer
electrodes?
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Direction of Transfer

• Perpendicularly from the direction of travel of
  proteins through the separating gel

Gel
Probe with specific Ab
Membrane
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Factors Affecting Transfer Efficiency

1. The Composition of the gel
2. Whether there is complete contact of the gel with
   the membrane
3. The position of the electrodes
4. The transfer time
5. The size composition of proteins
6. The field strength
7. The presence of detergents

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WB Procedure Briefly
1
2
4
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www.bio.davidson.edu/.../method/Westernblot.html
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Direct Detection Method
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Indirect Detection Method
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WB examples of used substrates
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Chemiluminescent substrates
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Enhanced ChemiFluoresenct (ECF) WB Detection
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Western Blotting Procedure Illustrated
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Steps of WB
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Steps of WB
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Steps of WB
Why to block? To increase sensitivity To prevent
nonspecific signal
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• Blocking of Blot
• Several measures should be followed to decrease
  the nonspecific reactions to a minimum, i.e.,
  increasing the signal to noise ratio.
• Blocking step is the incubation of the
  membrane with solution containing BSA or
  fat-free milk or casein for a sufficient time
  with shaking.

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Steps of WB
For Direct Transfer, choices are
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• Primary Antibody labeling
• The immobilized proteins on the surface of the
  membrane can be detected using a specific,
  labeled antibody.
• Labeling of the antibody can be performed using
  a radioactive or non- radioactive method.

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• Primary Antibody probing
• The blot is first incubated with a primary
  antibody followed by the addition of a labeled
  secondary antibody that has species specificity
  for the primary one.
• For example, probing of the membrane using
  mouse primary antibody and anti- mouse secondary
  antibody.

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Steps of WB
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Steps of WB
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• Detection and interpretation
• A prestained MW standard is included in a
  separate lane during electrophoresis to allow
  the identification of the MW of the target
  protein.
• Similar to the analysis of electrophoresis
  results on a gel, the data on the membrane can
  be quantitatively analyzed using gel
  documentation system.

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• Detection and interpretation (continue)
• Quantification of a specific protein band can
  be achieved by densitometry and integrating the
  areas under the peaks.
• Several gel documentation systems are
  commercially available that can be useful for
  analysis of results from the gel or membranes.

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Comparison between WB SB.

• Similarities
• Electrophoretically separated components
  (proteins in WB DNA in SB), are transferred
  from a gel to a solid support and probed with
  reagents that are specific for particular
  sequences of AA (WB) or nucleotides (SB).

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Comparison between WB SB, Contnd

• Differences
• The critical difference between SB WB is the
  nature of the probes

In WB
In SB
Probes usually are Ab(s) that react specifically
with Ag-ic determinants (epitopes) displayed by
the target protein
NA probes hybridize with a specificity rate
that can be predicted by simple equations,
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References

• Lippincott, Illustrated review of Biochemistry,
  4th edition
• Molecular Cloning A Laboratory Manual, J
  Sambrook, EF Fritsch, T Maniatis
• Catalogues of some commercial companies

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THANK YOU