Pharmaceutical biotechnology ..in that different blotting techniques such as ELISA , western blotting and southern blotting.There applications and their advantage and disadvantage with their diagrams

1. - Presented By Snehal D. Darekar
T.Y.B.Pharm
Roll no.13
Parikrama college of Pharmacy
Kashti.

3.  The Western blot is an analytical technique
used to detect specific proteins in a given
sample of tissue homogenate or extract.
 Immunoblotting: because an antibody is used
to specifically detect its antigen
 Western blot for Proteins was Developed by
George Stark using antibodies to locate
Proteins

4.  Western blotting is based on building
antibody-protein complex through specific
binding of antibodies to proteins immobilized
on a membrane.
 Then detecting the bound antibody through
several detection methods.

5.  Sample preparation
 Gel electrophoresis
 Transfer
 Blocking
 Antibody probing
 Detection
 Analysis

6.  • Protein from all sources can be used for
western blotting such as from single cell, whole
tissues, extracellular matrices, biological fluids.
 • Protein can be extracted from the sample in
three distinct steps:
 • Cell lysis: Physical method (ultra sonication,
homogenization etc.) Chemical method (use of
detergents) Biological method (enzymatic
digestion)
 • Protein isolation from the rest of cellular
components: Centrifugation, DNase and other
lysis buffer is used for the removal of cell
organelles, nucleic acids, polysaccharides etc
•Protein purification:

7.  • PAGE (Polyacrylamide gel electrophoresis) is
mainly used for the separation of protein
particles.
 • SDS is added to the gel and running buffer to
denature the protein molecules which will
ensure that your proteins are separated solely on
the basis of size and not on three-dimensional
structure.
 • Molecular weight markers are used to define
the size of proteins run in a gel. Markers are
composed of different proteins of known size.
 • As proteins are not directly visible in the gel,
the gel must be stained. Proteins are usually
stained with dyes such as Blue, Silver stain, or
Deep Purple.

8.  • Protein are transferred from the gel to a solid
support membrane such as nitrocellulose or
polyvinylidene difluoride (PVDF).
 • The proteins transferred from the gels are
immobilized which makes it possible to detect
the proteins on the membrane using specific
antibodies.
 • Electro-blotting is mainly used which uses
electric field to pull proteins from the gel onto
the PVDF or nitrocellulose membrane.
 • The proteins move from within the gel onto the
membrane while maintaining the organization
they had within the gel

10.  • Western blotting involves the
immobilization of biomolecules on a
membrane and blocking the spaces not
already occupied by proteins.
 • As non-specific binding of antibodies to the
membrane reduces the specificity and
sensitivity of the assay.
 • Two main classes of blocking agents
(proteins and non-ionic detergents) are
commonly used for western blotting.

11.  • Western blotting protocols usually uses a
non-labeled primary antibody directed
against the target protein and labeled
secondary antibody directed against the
constant region of the primary antibody.
 • The secondary antibody serves not only as
a carrier of the label but is also a mechanism
to amplify the emitted signals because
secondary antibodies are most often
polyclonal and can bind primary antibody at
different epitopes simultaneously.

12.  Different detection system are used in
western blotting based on fluorescence,
chromogenic or radio-isotopic detection.
 Enzymatic detection methods require the
addition of a substrate that emits light when
it reacts with an enzyme conjugated to a
secondary antibody.
 Fluorescence-based detection requires the
excitation of fluorophore using a light source
of a specific wavelength causing light
emission.

13.  • Diagnosis of HIV by ELISA involves the western
blotting technique.
 • Western blotting technique is also used to
detect some forms of Lyme Diseases.
 • Western blotting technique is used in definitive
test for BSE.
 • Confirmatory test for Hepatitis-B involves
western blotting technique.
 • Western blotting test is used in the analysis of
Biomarkers such as hormones, growth factors
and cytokines.
 • This technique is also employed in The Gene
expression studies.

14.  The enzyme-linked immuno sorbent assay
(ELISA) is an assay technique designed for
detecting and quantifying peptides, proteins,
antibodies and hormones.
 • The ELISA has been used as a diagnostic tool in
medicine and plant pathology, as well as a
quality-control check in various industries.
 • The process of the ELISA result in a colored end
product which correlates to the amount of
analyte present in the original sample.
 • ELISAs are typically performed in 96 well (or
384 well) polystyrene plates.

16.  • Principle of Enzyme-linked Immunosorbent
Assays (ELISAs) is based on the specific
interaction between antibody and antigen.
 • The ELISA can be used to detect antigens
that are recognized by an antibody or it can
be used to detect antibodies that are
recognized by an antigen.

18.  • All ELISA formats rely on the specific
interaction between an antigen and a
matching antibody.
 • The antibodies used in an ELISA can be
either:
 • monoclonal (capable of specific binding to
a single unique epitope)
 OR
 • polyclonal (capable of binding to multiple
epitopes).

21.  • A blocking buffer is a solution of irrelevant
protein, with a small percentage of
detergent. Blocking buffer block the places
where target proteins have not been
attached.
 • Washing buffer are used to remove
nonspecifically bound and unbound reagents.
 • Washing is performed by buffers such as
Tris-buffered saline (TBS) or phosphate-
buffered saline (PBS) with detergent such as
0.05% Tween20

23.  • Antigen is added to plate.
 • Added Blocking buffer.
 • Suitable primary antibody is added.
 • Secondary antibody- HRPO is then added
which recognizes and binds to primary
antibody.
 • TMB substrate is added, is converted to
detectable form.

25.  Apply a sample of known antigen to a
surface.
 • Enzyme linked primary antibody is applied
to the plate.
 • Wash the plate. After this wash, only the
antibody-antigen complexes remain
attached.
 • Apply a substrate which is converted by the
enzyme to elicit a chromogenic signal.

29.  • Presence of antigen or the presence of
antibody in a sample can be detected by ELISA.
 • ELISA can be used to determination of serum
antibody concentrations in a virus test (such as
HIV test).
 • ELISA can also be used in home pregnancy test.
• It is used in food industry to detect potential
food allergens such as milk, peanuts, walnuts,
almonds, and eggs.
 • ELISA can also be used in toxicology as a rapid
presumptive screen for certain classes of drugs.

30.  A Southern blot is a method used in
molecular biology for detection of a specific
DNA sequence in DNA samples.
 Southern blotting combines transfer of
electrophoresis -separated DNA fragments to
a filter membrane and subsequent fragment
detection by probe hybridization.
 The method is named after its inventor, the
British biologist Edwin Mellor Southern.

31.  The key to this method is hybridization.
Hybridization: It is the process of forming a
doublestranded DNA molecule between a single-
stranded DNA probe and a single-stranded target
DNA.
 There are 2 important features of hybridization:
 The reactions are specific-the probes will only
bind to targets with a complementary sequence.
 The probe can find one molecule of target in a
mixture of millions of related but non-
complementary molecules.

32.  1. Extract and purify DNA from cells;
 2. DNA is restricted with enzymes;
 3. Separated by electrophoresis;
 4. Denature DNA;
 5. Transfer to nitrocellulose paper;
 6. Add labeled probe for hybridization to
take place;
 7. Wash off unbound probe;
 8. Autoradiograph.

41.  To identify specific DNA in a DNA sample.
 To Isolate desired DNA for construction of rDNA.
 Identify mutations, deletions, and gene
rearrangements.
 Used in prognosis of cancer and in prenatal
diagnosis of genetic diseases.
 In RFLP.
 Diagnosis of HIV-1 and infectious disease.
 In DNA fingerprinting: Paternity and Maternity
Testing
 Criminal Identification and Forensics
 Personal Identification