About western blotting.

1. PRESENTATION ON
Applications of Western Blotting
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2. Overview
• Introduction
• Procedure
• Troubleshoot
• Applications
• Protein-protein interaction
• Future prospects
• Conclusion
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3. Introduction
Western blotting is a technique for detection of
the presence of a particular piece of protein in a
sample of tissue homogenate or extract, also
called immune-blotting.
It was introduced by Towbin, et al. in 1979 and
is now a routine technique for protein analysis,
clinical research, life science research.
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4. Sample preparation
Gel Electrophoresis
Sample transferred to membrane
Blocking
Incubation/Antibody Probing
Detection
Analysis
Western blotting procedure
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Flow chart of western blotting procedure

5. Troubleshooting
Even though the procedure for western blot is
simple, many problems can arise, leading to
unexpected results. The problem can be grouped
into five categories:
Unusual or unexpected bands
No bands
Faint bands or weak signal
High background on the blot and
Patchy or uneven spots on the blot.
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6. Detects proteins and estimates their
molecular weight
Samples containing protein targets are prepared
normally.
The samples are mixed with dithiothreitol.
Then loaded on a Western assay plate with wash
buffers, chemiluminescence substrate, primary and
secondary antibodies.
Sample placed into the Western blot system with the
appropriate capillary set and the run is started.
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7. Detects proteins and estimates their
molecular weight
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Source: http://www.bio-rad.com/en-us/applications-technologies/introduction-
western-blot-protein-standards
Figure 01: Detection of molecular weight of sample.

8. Analysis of IgG fractions purified from
human plasma
Plasma contains a large variety of proteins including
immunoglobulins, used in therapeutic applications,
such as passive prophylaxis.
Purification of the plasma proteins, transferrin and
fibrinogen, were also identified by Western blotting.
Samples from the different purification steps were
separated on a 4% to 20% gradient Amersham ECL
(enhanced chemiluminescence) Gel and stained
using Deep Purple Total Protein Stain.
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9. Analysis of IgG fractions purified from
human plasma
Figure 02: Plasma fractions in an IgG purification process stained using Deep Purple
Total Protein Stain. The enrichment and purification of IgG can be followed through
different steps from the original plasma pool (1) to purified IgG heavy chain (14).
Fractions run in lanes 8 and 11 show where albumin and transferrin, respectively, are
separated from IgG.
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Source: https://www.sigmaaldrich.com/content/ge-western-blotting

10. Confirmatory HIV-test
 The WB is the most widely accepted confirmatory assay
for the detection of antibodies to the retroviruses.
 A HIV antigen mixture is layered onto a sodium dodecyl
sulfate (SDS) polyacrylamide gel slab and then electro-
phoresed. The viral proteins (HIV antigens) migrate
through the molecular pores of the gel at rates determined
by electrical charge and molecular weight.
 Reaction with a positive serum sample produces a pattern
of bands on the strip that is characteristic of HIV. Many of
these bands have been identified as specific viral gene
products.
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11. Confirmatory HIV-test
Figure 03: The HIV-1 viral antigens are separated as follows (from top to bottom):
gp160, gp120, p68, p55, p51, gp41, p40, p34, p24, and p17. The "gp" designation
refers to glycoproteins; "p" indicates proteins. The numeric values (x100) indicate
molecular weights. 11
Source: https://microbeonline.com/laboratory-diagnosis-of-hiv-virus/

12. Western Blot Diagnosis of Hepatitis B virus
 The genome of hepatitis B virus (HBV) is a double-
stranded DNA molecule within virus particles that is
approx 3.2 kbp long.
 Tissues and cells were lysed in lysis buffer containing a
protease inhibitor cocktail. Cell extract protein amounts
were quantified using the protein assay.
 Primary antibodies: Hepatitis B Virus X protein(HBX),
stress-activated protein kinase, phosphostress activated
protein kinase, and β-actin. Also add Secondary antibodies
and ECL solutions.
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13. Western Blot Diagnosis of Hepatitis B virus
Figure 04: Western blotting for HBX proteins in specimens. HBX proteins expression
level was higher in ulcers than matched normal tissues (P<0.05). N: normal, U: ulcer. β-
actin was used as an internal control. 13
Source: https://www.spandidos-publications.com/or/28/5/1653

14. Protein-protein interaction
Far western blotting (WB) was derived from the
standard WB method to detect protein–protein
interactions in vitro.
Far WB determines whether two proteins bind to
each other directly or indirectly, Far WB allows the
examination of candidate protein(s) that form a
complex between them. Typically, 2–3ds are required
to carry out the experiment.
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15. Future prospects
Western blotting is a widely used technique but it is
time consuming so researcher should establish less
time consuming technique.
If proteins are degraded quickly it cant detect the
protein so modification should necessary in this
technique.
The materials used in this technique are costly so
need to identified cheap materials for large scale
identification.
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16. Conclusion
Western blot is a technique that is useful for protein
detection as it allows the user to quantify the protein
expression as well.
It’s a powerful and indispensable scientific techniq-
ue that can be used to accurately quantify relative
protein levels and necessary for proper experimental
techniques and strategies are employed.
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17. Thanks To All For Your Kind
Attention.
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