In this slide contains principle, types, methods and application of Western Blotting Technique. Presented by: T.NIRANJAN REDDY (Department of pharmacology). RIPER, anantapur

1. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement for
M . Pharmacy I year I Semester
Presented by
T. Niranjan Reddy
(20L81S0109)
Department of Pharmacology
Under the guidance of
Dr. P. Ramalingam, MPharm, Ph. D
Research Director and professor of Pharmaceutical and medicinal
chemistry
WESTERN BLOTTING

2. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Contents :
 Introduction
 Types of blotting techniques
 Principle of western blotting
 Procedure for western blotting
 Gel electrophoresis
 Protein transfer
 Antibody probing
 Protein detection
 Analysis and imaging
 Applications
 Limitations
2

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Introduction:
Blotting
 Blotting is a method of transferring proteins, DNA or
RNA, on to a carrier (for example, a nitrocellulose or
PVDF or nylon membrane)
3

4. RIPER
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NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721

5. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
 Western blotting is a widely used analytical technique in molecular
biology to detect specific protein in a sample of tissue homogenate
or the extract
 It works on the principle of gel electrophoresis
 Proteins are separated based on their size on polyacrylamide gel
Western blotting

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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
• Western blotting is an immunoblotting technique which rely on the
specificity of bonding between a molecule of interest and a probe to
allow detection of the molecule of interest in a mixture of many
other similar molecules
• In western blotting, the molecule of interest is a protein and the
probe is typically an antibody raised against that particular protein
• The SDS PAGE (sodium dodecyl sulphate –polyacrylamide gel )
technique is a prerequisite for western blotting.
Principle of western blotting

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7

8. RIPER
AUTONOMOUS
NAAC &
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8

9. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• The proteins of the sample are separated by using the gel
electrophoresis
• Electrophoresis is a commonly used method for separating proteins
on the basis of size, shape and charge
• In SDS (sodium dodecyl sulphate) electrophoresis, protein samples
are separated according to their molecular weight.
Gel electrophoresis

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Page protocol

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Sample Loading
11

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 On completion of the separation of proteins by polyacrylamide gel
electrophoresis, the next step is to transfer the proteins from the gels to
solid support membrane
 This solid support membrane usually made up of a chemically inert
substance such as nitrocellulose or PVDF (polyvinylidene difluoride)
 The process of transferring proteins from a gel to a membrane while
maintaining their relative positions and resolutions is known as blotting.
protein transfer:

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 After gel electrophoresis it may be necessary to confirm that all the
proteins in the gel have been completely eluted
 As proteins are not directly visible in the gel, the gel must be stained
 Proteins are usually stained with dyes such as Coomassie blue, sliver
stain or deep purple
 After staining a permanent record may be made by imaging the gel
with the suitable instrument.
Protein staining:

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15. RIPER
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Blocking :
 The membrane has the ability to bind to proteins in this case
both the target and antibodies are proteins and so there could
be some unwanted binding.
 For meaningful results, 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 inert protein or non-ionic
protein
 Blocking agents should possess greater affinity towards
membrane than the antibodies
15

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The most commonly used blocking agents are :
 Bovine serum albumin (BSA)
 Non –fat milk
 Casein
 Gelatine
 Dilute solution of tween 20
Blocking agents

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Antibody probing
 After blocking, the blot is incubated with one or more antibodies.
 This uses specific antibody to detect a localize the protein blotted to the
membrane
 The specificity of antigen antibody permits the identification of a single protein
in a complex sample
 The non-labelled primary antibody directed against the target protein and
specific labelled secondary antibody binds to primary antibody
 The secondary antibody is conjugated to an enzyme that is used to indicate the
location of protein
 Secondary antibodies can be a monoclonal or polyclonal antibodies
17

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Washing
• The unbound antibodies can cause high background and poor
detection
• Hence, washing the blot removes unbound antibodies from the
membrane
• A dilute solution of tween – 20 in TBS or PBS buffer is
commonly used for washing
18

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Protein detection
• After the unbound probes are washed away, the western
blotting is now ready for detection of probes that are labelled
and bound to the protein of interest
• The marked antibody which is linked to a reporter enzyme
which when exposed to appropriate substrate drives a
colorimetric reaction and produces a colour
• Enzymes such as alkaline phosphatase(AP) and horse radish
peroxidase (HRP) are widely used in detection of proteins

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
 This is the last and major step of the western blotting technique
 Detection of signals using either x-ray film, scanners or a CCD,
results in one or more visible proteins on the membrane image
 The molecular weight of protein can be estimated by comparison
with marker proteins and the amount of protein can be determined
and this is related to band intensity
 Qualitative and quantitative analysis can be done in order to verify
the absence or presence of specific proteins of interest.
Analysis and imaging

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 Analysis of igG fractions purifies from human plasma
 Diagnosis of HIV by ELISA, involves the western blotting technique
 Western blotting technique is also used to detect some forms of
Lyme disease
 Western blotting technique is used in definitive test for BSE, which
is commonly known as mad cow disease
 confirmatory test for hepatitis-B involves western blotting
technique
 This technique are employed in the gene expression studies.
Application of Western Blotting

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 Very delicate and time consuming process
 If a protein is degraded quickly, then western blotting technique
wont detect it well
 Well trained techniques are required for this technique
 Primary antibody availability is crucial.
Limitations of Western Blotting

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References :
• Kurien BT, Scofield RH. 2006. Western blotting. Methods 38: 283–
293.
• Jensen EC. The Basics of Western Blotting. Anat Rec. 2012
Mar 1;295(3):369–71.
• Antharavally BS, et al. A high-affinity reversible protein stain
for Western blots. Analytical biochemistry. 2004;329(2):276–
80
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K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25