Blotting Techniques

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Blotting Techniques
Blotting Techniques
Mazen Al Zaharna MSc Biological Sciences- Medical
Technology Medical Technology Dep. The Islamic
University- Gaza
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Introduction

• Definition
• A technique by which a macromolecule such as DNA,
  RNA, or protein is resolved in a gel matrix,
  transferred to a solid support, and detected with
  a specific probe.
• Used to identify specific molecules in a complex
  mixture of related molecules.

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Introduction

• Common techniques include
• Southern blotting (DNA) ,
• Northern blotting (RNA) ,
• and immunoblotting (for protein also known as
  Western blotting).

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• The blotting procedures can be divided into six
  main steps

1- Electrophoresis
2- Transfer
3- Blocking
4- Probing
5- Detection
6- Results
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1- Electrophoresis

• The molecule of interest is present in a complex
  mixture of molecules.
• Separate on the basis of size.
• Separating the molecules by gel electrophoresis
  on either
• an agarose
• or polyacrylamide gel.

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Gel Electrophoresis
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2- Transfer (blotting)

• Following separation, the molecules are
  transferred to a solid support such as
• a nylon,
• nitrocellulose,
• or polyvinylidene fluoride (PVDF) membrane.
• Carbon copy of the molecules that were present in
  the gel are now immobilized on a membrane.

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The membrane of Choice
Type of Membrane
The membrane of choice is determined by the
sensitivity required and the detection method to
be used.
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2- Transfer (blotting)- Blotting Types
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2- Transfer (blotting)- Capillary Transfer

• Fragments are eluted from the gel and deposited
  onto the membrane by buffer that is drawn through
  the gel by capillary action.

Paper towel stack
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2- Transfer (blotting)- Electrophoretic Transfer

• The negatively charged nucleic acid molecules
  will move from the gel to the membrane

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Electroblotting
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2- Transfer (blotting)- Vacuum Transfer

• Nucleic acids are eluted by buffer that is drawn
  through the gel by application of negative
  pressure (a vacuum).

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2- Transfer (blotting)- Cross-Linking

• Once transferred to a membrane, they have to be
  linked to the membrane.
• UV irradiation, covalently attach the nucleic
  acids to the membrane
• Covalent bond between the amide groups on the
  nylon and the carbonyl groups found on the
  thymine and uracil bases

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2- Transfer (blotting)- Cross-Linking

• Alternatively, the membrane can be baked at 80?C
  for 2 hr.
• Dehydration of the nucleic acids on the blot,
• resulting in the generation of stable hydrophobic
  interactions between the nucleic acid and the
  membrane.

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3- Prehybridization (Blocking)

• The transferred nucleic acids only occupy a
  limited amount of the surface area of the
  membrane.
• The molecules in the prehybridization solution
  coat the rest of the membrane.
• In the absence of such a treatment, the probe
  would
• associate with the unoccupied sites on the
  membrane,
• resulting in very high background and a very low
  signal-to-noise ratio.

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4- Probing

• Membrane is now incubated with a specific probe
  that binds to the protein or nucleic acid
  sequence of interest.
• For southern or northern, a fragment of DNA of
  variable length (usually 100-1000 bases long)

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4- Probing

• The probe will have two properties
• First, anneal specifically with the sequence of
  interest.
• Second, modified in such a way as to allow for
  the detection of the annealed sequences.

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4- Probing

• Probe used for an immunoblot is an antibody that
  recognizes a particular protein
• 2o Ab with a label will bind to 1o Ab with high
  affinity
• Unbound probe or nonspecifically bound probe is
  removed by washing the membrane

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4- Probing
Probe
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4- Probing- Production of Probes

• The availability of a gene probe is essential in
  many molecular biology techniques.
• The information needed to produce a gene probe
  may come from many sources,
• e.g. genetic databases.
• Genbank and EMBL search to identify particular
  sequences relating to a specific gene or protein.

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4- Probing- Production of Probes

• Use related proteins from the same gene family to
  gain information DNA sequence.
• Similar proteins or DNA sequences but from
  different species may also provide a starting
  point with which to produce a probe.

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4- Probing- Labeling of Probes

• To visualize DNA or RNA, the nucleic acid should
  to be attached to a label
• radioactive,
• colored,
• fluorescent,
• Or luminescent.
• The three main choices are
• radioisotopes,
• fluorophores,
• and small-molecule binding partners

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4- Probing- Labeling of Probes- A- Radioisotopes

• 32P is commonly used as a label
• Emits radiation that can be easily detected by
  autoradiography
• Nucleotides that incorporate 32P are commercially
  available.
• Can be readily incorporated into DNA by
  enzyme-catalyzed reactions.

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4- Probing- Labeling of Probes- B- Fluorophores

• Fluorophores are molecules that absorb light at
  one wavelength and then emit light at a different
  wavelength.
• Incorporate fluorophores
• chemically during DNA synthesis,
• or enzymatically

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4- Probing- Labeling of Probes- C- Small
molecule binding partners

• Small organic molecules that are recognized by
• antibodies or
• other protein binding partners.
• Common molecules are biotin and digoxigenin

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5- Detection

• Streptavidin covalently conjugated with a
  detection moiety.
• For example, streptavidin conjugated directly to
  a fluorophore or to enzymes such as horseradish
  peroxidase or alkaline phosphatase.
• Enzymes detected by their action on provided
  substrates that deposit products which are
  colored, luminescent, or fluorescent.

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4- Probing- Labeling of Probes- C- Small
molecule binding partners
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Membrane
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Hybridization

• Probe is generated and added to the blot for 1 to
  24 hr.
• Time to hybridize the blot depends on a variety
  of factors and must be determined empirically.
• Overnight to maximize hybridization of the probe
  to the target

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5- Detection

• To visualize the bound probe.
• Determined by the nature of the probe.
• If a radioactive probe, autoradiography
• exposure of the blot to X-ray film will allow for
  detection and quantitation of the bound probe.

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5- Detection

• If chemical- or enzyme-based,
• substrates are added
• the resulting signal is developed
• and can be documented by
• colorimetric,
• or chemiluminescent imaging.

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5- Detection

• For fluorescently labeled nucleic acid
• use imaging equipment to excite the fluorophore
• And the appropriate filter to detect the emitted
  light.

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6- Results and Analysis

• Once the blot is developed, the resulting banding
  pattern can be analyzed.
• Analysis involves
• determining the amount and molecular weight or
  size of the molecules on the blot
• and comparing the results to the predicted
  pattern.
• To determine the molecular weight a standard
  curve of size versus migration distance is
  derived from the molecular weight markers

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3
Best Fit Line
2
Log- Molecular Weight
1
Distance (mm)
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Positive Negative Controls

• Negative controls
• Include samples that are identical to the
  experimental sample but are missing the target
  that the probe is supposed to recognize.
• Very useful in determining the existence of any
  background that can be due to cross-reactivity
  between the probe and the sample.

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Positive Negative Controls

• Positive control
• Include samples that contain the protein or
  nucleic acid of interest.
• When included in the experiment allows the
  investigator to confirm that the experiment was
  successfully executed.
• No signal indicates that the problem lies with
  the experimental samples and not with the
  procedure.

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Southern Blotting
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Southern Blotting

• Developed by E.M. Southern in 1975.
• A technique used in molecular biology to check
  for the presence of a particular DNA sequence in
  a DNA sample.

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Flow chart of Southern hybridization

• Preparing the samples and running the gel
• Southern transfer Fixing DNA onto membrane
• Probe preparation
• Prehybridization
• Hybridization
• Post-hybridization washing
• Signal detection

Isotope Non-isotope
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Preparing the samples and running the gel

• Extraction of DNA
• DNA must first be fragmented into small pieces
  that can migrate through an agarose gel matrix.
• Restriction enzymes are used to fragment the DNA

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Preparing the samples and running the gelDNA
Digestion

• Restriction enzymes recognize specific DNA
  sequences in DNA and cleave the DNA at these
  restriction sites.
• Digestion with a given restriction enzyme
  produces a set of fragments that are easily
  separated by agarose gel electrophoresis.

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Preparing the samples and running the gelDNA
Digestion
The enzyme EcoRI cutting DNA at its recognition
sequence
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Preparing the samples and running the gel
Electrophoresis

• Nucleic acids are negatively charged at a neutral
  pH
• This allows their migration through an electric
  field
• Agarose is a highly porous polysaccharide that
  acts as a sieve, allowing the fragments of DNA to
  be separated according to length.

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Preparing the samples and running the gel
Electrophoresis
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Preparing the samples and running the
gelDenature the DNA

• Denature DNA with an alkaline solution such as
  NaOH.
• Double stranded becomes single-stranded.
• Single strands are ready to be transferred to a
  solid support

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Southern Transfer Fixing DNA

• Transfer the DNA from the gel to a solid support.
• Baking the membrane at 80C for 2 h in a vacuum
  oven.
• Or expose to ultraviolet

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Probe Preparation, Prehybridization
Hybridization

• A labeled probe is prepared which is
  complementary for the sequence we are looking for
• Prehybridization to block sites where probe can
  bind on the membrane
• Hybridization

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Post-hybridization washing

• Following hybridization, the blot must be washed
  to remove unassociated and nonspecifically
  annealed probe from the blot.

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Detection
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Steps in Southern Blotting
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Uses of Southern Blotting Technique

• Identify mutations, deletions, or rearrangements
  that alter the integrity of a specific gene,
• useful in the prognosis of certain types of
  cancer
• Tool for molecular cloning, providing a mechanism
  for localization of specific sequences

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Uses of Southern Blotting Technique

• The DNA blot can also be used to assess the
  relative copy number of a specific gene.
• Useful in detecting gene amplification.
• Southern blotting may be used to confirm the
  specificity of the test reaction product.
• To search for a homologous gene different
  organisms

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Southern Blotting as a Diagnostic Method

• Restriction fragment length polymorphism (RFLP)
  analysis was one of the early methods to diagnose
  point mutations implicated in genetic diseases
• The change in the size of detected fragments with
  a gene-specific probe signals the presence of
  mutation in the analyzed gene
• Has been applied to the diagnosis of hemophilia
  A, Sickle cell anemia and others

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Southern Blotting as a Diagnostic Method

• PCR has replaced the Southern blotting
• Cystic fibrosis, Duchenne muscular dystrophy,
  sickle cell anemia thalassaemia, and others, are
  now diagnosed by polymerase chain reaction (PCR).

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Genomic and Plasmid DNA Analyses

• Does a particular genomic locus or region of
  plasmid DNA contain a sequence of interest? Where
  does it reside?
• Techniques
• Restriction enzyme digestion
• Agarose gel electrophoresis
• Southern blot

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Genomic and Plasmid DNA Analyses

• How many genomic loci contain a particular
  sequence of interest, or how many copies of that
  sequence does a genome contain?
• Technique
• Southern blot

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The Northern Blot
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The flow chart of Northern hybridization

• Prepare RNA samples and run RNA gel
• Northern transfer
• Probe preparation
• Prehybridization
• Hybridization
• Post-hybridization washing
• Signal detection

Isotope Non-isotope
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• Allows identification of specific messenger RNA
  sequences within a mixture of RNA molecules.
• The final signal achieved on the blot is
  proportional to the number of specific sequences
  present,
• allowing for a quantitative analysis of gene
  expression.

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Differences between Southern Northern

• RNA rather than DNA is separated by size on gel
• Cutting by nucleases before electrophoresis
  unnecessary.
• Although RNA is single stranded, it has a
  tendency to bend back on itself and form
  base-paired loops, hairpins, and other secondary
  structures.
• Denaturing agents (e.g., formamide) must be added
  to the electrophoresis buffer to prevent the
  formation of secondary structures

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Differences between Southern Northern
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RNA Paranoia

• RNA paranoia is very important from start to
  finish.
• The work area should be cleaned with RNase
  inhibitors.
• Gloves should be changed if non-RNase-free items
  have been touched (e.g., your hair, your face,
  your arm, notebook paper).

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Uses of Northern Blotting

• Northern blots can be used to assess different
  levels of expression from a particular gene.
• For defining post-transcriptional modification
  such as
• splicing and poly(A) addition,

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Gene Expression (Transcription) Analyses

• What is the size of a specific gene transcript?
• Technique
• Northern blot

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Gene Expression (Transcription) Analyses

• Is a gene of interest expressed (transcribed)?
• Technique
• Northern blot

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Gene Expression (Transcription) Analyses

• Is transcription of a gene altered (increased or
  decreased) under different conditions?
• Technique
• Northern blot
• Real-time PCR (for more quantitative comparison)

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Immunoblotting(Western Blotting)
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The Flow Chart Of Immunoblotting

• Electrophorese samples
• Transfer proteins from gel to membrane
• Blocking
• Addition of 1o Ab, washing
• Addition of 2o Ab, washing
• Detection

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Uses of Immunoblotting

• Immunoblotting is used to identify specific
  protein in a mixture

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Uses of Immunoblotting
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Dot and slot blots

• Provide a quick and simple way to determine the
  amount of an antigen in a sample without
  performing electrophoresis first.
• Proteins are deposited onto the membrane
• Probe with the same chromogenic or luminescence
  protocol as the western blot.

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Dot and slot blots

• Provides a mean of measuring the abundance of
  specific proteins without the need for gel
  electrophoresis,
• It does not, however, provide information
  regarding the size of the fragments.

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Proteins

• In which cellular structures or organelles do
  specific proteins reside?
• Techniques
• Cell fractionation
• Immunoblotting

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Proteins

• What is the molecular mass of a specific protein?
  Is it post-translationally modified?
• Technique
• Immunoblotting

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Example for Uses of Blotting Techniques

• Suppose a student was studying a newly identified
  gene, X, from cows.
• The student then asks three basic questions as
  part of a research project
• Do sheep also have gene X on their chromosomes?
• Do cows express gene X in their brain tissue?
• Is the protein product of gene X found in the
  cow's blood plasma?
• Blotting experiments can answer all three of
  these questions.

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Do sheep also have gene X on their chromosomes?

• A Southern (DNA) blot will answer the first
  question.
• DNA from a sheep and performed the Southern
  blotting technique with a probe complementary to
  that gene.
• If the sheep's DNA also contains gene X, there
  should be a fragment on the nitrocellulose
• In other words, the labeled probe will bind to
  any fragment from the blotted sheep DNA that
  contains gene X, allowing the student to detect
  the presence of gene X in sheep.

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Do cows express gene X in their brain tissue?

• To answer the second question, a Northern (RNA)
  blot would be used.
• The student would isolate RNA from the cow's
  brain tissue and run it out on the gel.
• The same DNA probe used for Southern would then
  be used to detect whether the RNA that represents
  gene X expression is present in the brain.

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Is the protein product of gene X found in the
cow's blood plasma?

• To answer the third question, the student would
  use a Western (protein) blot.
• This requires the use of an antibody that
  specifically reacts with the protein coded for by
  gene X.
• The student first obtains plasma from the cow and
  uses standard biochemical techniques to isolate
  the proteins for analysis.

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• These proteins can then be run out on a gel and
  transferred to nitrocellulose.
• The proteins can then be probed with the labeled
  antibody.
• If the product of gene X is in the plasma, it
  will bind with the labeled antibody and can thus
  be detected.

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References

• Current Protocols Essential Laboratory Techniques
  (2008)
• Molecular Diagnostics (2006)
• Medical Biomethods Handbook (2005)

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Thank You