Western Blotting and
its Applications
Bachelor of Education Biological Sciences
Batch 7
Molecular Biology and its Application
(BDB32081E)
Student Id: BEd 72018172
Course Instructor : Dr. Roshini Wimalarathna
CONTENT
1. Introduction
2. Blotting Techniques
3. Procedure
4. Applications
5. Limitations
6. References
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INTRODUCTION
Blot refers to membrane.
Blotting are techniques for
transferring DNA , RNA
and proteins onto a carrier
so they can be separated,
and often
follows the use of a gel
electrophoresis in
detection.
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TYPES OF BLOTTING TECHNIQUES
Blotting Technique
Southern Blot
Detect DNA
Edwin Southern
(1997)
Northern Blot
Detect RNA
James Alwine, Dave
Kemp and George
Stark (1997)
Western Blot
Detect protein
Western Neal Burnette
(1981)
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WESTERN BLOTTING
The Western blotting / protein blotting or immunoblotting is
- an analytical technique used to detect specific proteins
- separated by electrophoresis by use of labeled antibodies
- in a given sample of tissue homogenate or extract which
- rely on the specificity of binding between a molecule of
interest and a probe to allow detection.
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Principles of Western Blotting
• Analyze any protein sample from cells or tissues, and
recombinant proteins synthesized in vitro
• Based on protein - protein interaction or antigen - antibody
interaction
• Specificity of the binding to the target and low cross-reactivity
• Depends on 3 factors:
a- The ability of the antibody to bind a specific protein
b- The strength of the interaction
c- The concentration of the protein of interest itself
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PROCEDURE
CONTENT
Sample preparation
Gel Electrophoresis
Blotting or transferring
Blocking
Antibody Probing
Detection
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1. Sample Preparation
• Samples may be taken from whole tissue or from cell culture.
• Solid tissues are first broken down mechanically using a
blender.
• Bacteria, virus or environmental samples can be the source of
protein (Western blotting is not restricted to cellular studies.)
• Assorted detergents, salts, and buffers may be employed to
encourage lysis of cells and to solubilize proteins.
• Tissue preparation is often done at cold temperatures to
avoid protein denaturing.
• By applying various types of filtration and centrifugation
methods for further preparation for the samples.
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Sample Preparation
Detergent Lysis for
tissue culture
Ultra sonication
for cell
suspension
Mechanical
homogenization for
Plant, animal tissue
Enzymatic Digestion
for Bacterial, Yeast
and Fungal cells
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2. Gel Electrophoresis (SDS-PAGE)
• A technique in which charged molecules such as protein; separated into their
components according to physical properties as forced through a gel by an
electrical current to detect presence of a specific protein.
• Separation of proteins by isoelectric point, molecular weight, electric charge,
or a combination of these factors.
• Most widely used technique for protein electrophoresis is odium
ulfate oly crylamide el lectrophoresis (SDS-PAGE)
odecyl
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• This technique sets proteins
according to their molecular
weights
• All the separated proteins in the
gel have uniform (-) charge
because of sodium dodecyl
sulfate which coats the proteins
• So polyacrylamide gel after SDSPAGE, the band's shown
represents proteins present in
each sample
• The leftmost plane of the gel is
represent in the molecular
weight markers
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3. Blotting or transferring
 The process of transferring proteins from a gel to a membrane while
maintain their relative position and resolutions is known as blotting.
 Need: further detection and analysis of separated proteins.
 The membranes used in Western blotting are having high affinity for
proteins, excellent protein binding and retention capabilities.
 The membranes are thinner than gels therefore
when proteins bind to these membranes their
epitope saw binding sites are easily accessible to
the antibodies.
 To make the proteins accessible to antibody
detection, they are moved from within the gel
onto a membrane made of Nylon, nitrocellulose
or polyvinylidene difluoride (PVDF).
 As proteins are not directly visible in the gel they
usually stained with dyes such as coomassie blue,
silver stain or deep purple.
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a) Electrophoretic Transfer
•
•
•
The method used to transfer proteins from gel to membrane is known as
electrophoretic transfer.
Electric current is used to elude proteins from gel (- electrode side) and
transfer them to membranes (+ electrode side) that placed in the
electrophoresis chamber.
Proteins in the gel are negatively charged so they move out of the gel and
migrate towards the positive electrode.
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b) Transfer Sandwich
•
The membrane is placed on top of the gel, and a stack of filter papers
placed on top of that. The entire stack is placed in a buffer solution which
moves up the paper by capillary action, bringing the proteins with it.
•
Filter paper – maintains uniform flow of transfer buffer through gel
facilitating the movement of proteins
•
Sponge – maintains proper pressure during the transfer
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• Complete setup of sandwich of gel and membrane is held inside a nonconducting cassette kept entirely submerged under transfer buffer within a
buffer tank.
• Cassette placed as gel at (-) electrode side and membrane at (+) electrode
side. When electric current is applied the negatively charged proteins
travel from gel toward the membrane at the (+) electrode side.
• Methanol present in transfer buffer facilitates binding of proteins to the
membrane.
• All the proteins from gel moved to the membrane and become tightly
attached to it giving a membrane with copy of band pattern from gel
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4. Blocking
 For meaningful result, the antibodies must bind only to the protein of
interest and not to the membrane.
 Non-specific binding of antibodies can be reduced by blocking the
unoccupied sites of membrane with an inert protein or non-ionic detergent.
 Blocking agents should possess greater affinity towards membrane than the
antibodies.
 The most common blocking agents are:a) Bovine serum albumin (BSA)
b) Non-fat milk
c) Casein
d) Gelatine
e) Dilute solution of Tween 20
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5. Antibody probing
a) Primary Antibody probing
 After the blocking step the membrane is incubated with primary antibody.
 Since this antibody is specific to our target protein it will bind to the protein
on the membrane.
 Later the excess of primary antibodies are removed by washing.
Washing
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5. Antibody probing
b) Secondary Antibody probing
 The membrane is incubated with secondary antibody to maximize the
sensitivity of the detection.
 When the membrane is incubated with labeled secondary antibody (an
enzyme like Horseradish Peroxidase-HRP) that bind to the primary antibody
is already bound to the target protein on the membrane.
 Multiple secondary antibodies can bind to the target primary antibody and
this results in the amplification of detection signal.
 Then excessive secondary antibodies are also removed by washing.
Washing
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5. Detection
 Finally in the presence of HRP like enzymes act upon colorimetric or
chemiluminescence substrates.
 If the membrane is incubated with a colorimetric substrate such as 4choloro-1-naphtol the enzyme in the membrane catalyzes the oxidation of
the substrate into an insoluble purple product and this purple color is visible
on a blot which is the target protein.
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APPLICATIONS
To determine the size and amount of protein in given sample
Analyze IgG fractions purified from human plasma
Detect defective proteins when doing definitive test for Bovine
spongiform encephalopathy (Mad cow disease)
Disease diagnose by detecting antibody against virus or
bacteria in serum
 Some forms of Lyme disease testing employ
 The confirmatory HIV test detects anti HIV antibody in
patients serum
 Used as a confirmatory test for Hepatitis B infection and
Herpes.
In veterinary medicine, sometimes used to confirm FIV+
status in cats.
Used in gene expression studies
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LIMITATIONS
 If a protein is degraded quickly, Western blotting won't
detect it well.
 Incorrect labelling of protein can happen due to the
reaction of secondary antibody.
 Very delicate and time consuming process
 It might also be more costly
 Subject to interpretation
Presence or absence of bands
Intensity of those bands
 Well trained technicians are required for this technique.
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REFERENCES
 https://www.onlinebiologynotes.com/western-blotting-technique-principle-procedureapplication/
 https://blog.praxilabs.com/2020/05/29/western-blot-concept/
 https://youtu.be/CEEekahiqMo
 https://youtu.be/mjbr3beDslo
 https://youtu.be/Ll_7z4YS2Ak
 http://www.slideshare.net/vivekaiden/western-blotting 119614678
 http://www.slideshare.net/AshfaqAhmad52/western-blotting-87621064
 http://www.slideshare.net/AkanshGoel5/western-blotting-technique
 http://www.slideshare.net/indusahyadri96/western-blotting-power-point-presentation
 https://www.biotechniques.com/biochemistry/south-north-east-and-west-ern-the-story-ofhow-the-western-blot-came-intobeing/#:~:text=Burnette's%20western%20blot%20has%20since,invention%20of%20the%20
western%20blot.
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