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Blotting Techniques

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Title: Blotting Techniques


1
Blotting Techniques
Blotting Techniques
Mazen Al Zaharna MSc Biological Sciences- Medical
Technology Medical Technology Dep. The Islamic
University- Gaza
2
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.

3
Introduction
  • Common techniques include
  • Southern blotting (DNA) ,
  • Northern blotting (RNA) ,
  • and immunoblotting (for protein also known as
    Western blotting).

4
  • The blotting procedures can be divided into six
    main steps

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

6
Gel Electrophoresis
7
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.

8
The membrane of Choice
Type of Membrane
The membrane of choice is determined by the
sensitivity required and the detection method to
be used.
9
2- Transfer (blotting)- Blotting Types
10
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
11
2- Transfer (blotting)- Electrophoretic Transfer
  • The negatively charged nucleic acid molecules
    will move from the gel to the membrane

12
Electroblotting
13
2- Transfer (blotting)- Vacuum Transfer
  • Nucleic acids are eluted by buffer that is drawn
    through the gel by application of negative
    pressure (a vacuum).

14
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

15
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.

16
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.

17
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)

18
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

21
4- Probing
Probe
22
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.

23
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.

24
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

25
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.

26
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

27
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

28
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.

29
4- Probing- Labeling of Probes- C- Small
molecule binding partners
30
Membrane
31
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

32
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.

33
5- Detection
  • If chemical- or enzyme-based,
  • substrates are added
  • the resulting signal is developed
  • and can be documented by
  • colorimetric,
  • or chemiluminescent imaging.

34
5- Detection
  • For fluorescently labeled nucleic acid
  • use imaging equipment to excite the fluorophore
  • And the appropriate filter to detect the emitted
    light.

35
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

36
3
Best Fit Line
2
Log- Molecular Weight
1
Distance (mm)
37
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.

38
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.

39
Southern Blotting
40
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.

41
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
42
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

43
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.

44
Preparing the samples and running the gelDNA
Digestion
The enzyme EcoRI cutting DNA at its recognition
sequence
45

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.

46
Preparing the samples and running the gel
Electrophoresis
47
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

48
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

49
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

50
Post-hybridization washing
  • Following hybridization, the blot must be washed
    to remove unassociated and nonspecifically
    annealed probe from the blot.

51
Detection
52
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

56
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

59
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).

60
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

61
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

62
The Northern Blot
63
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
64
  • 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.

65
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

66
Differences between Southern Northern
67
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).

68
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,

69
Gene Expression (Transcription) Analyses
  • What is the size of a specific gene transcript?
  • Technique
  • Northern blot

70
Gene Expression (Transcription) Analyses
  • Is a gene of interest expressed (transcribed)?
  • Technique
  • Northern blot

71
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)

72
Immunoblotting(Western Blotting)
73
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

74
Uses of Immunoblotting
  • Immunoblotting is used to identify specific
    protein in a mixture

75
Uses of Immunoblotting
76
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.

77
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

80
Proteins
  • What is the molecular mass of a specific protein?
    Is it post-translationally modified?
  • Technique
  • Immunoblotting

81
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.

82
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.

83
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.

84
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.

85
  • 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.

86
References
  • Current Protocols Essential Laboratory Techniques
    (2008)
  • Molecular Diagnostics (2006)
  • Medical Biomethods Handbook (2005)

87
Thank You
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