B’ PHARMACY

SUBJECT-PHARMACEUTICAL BIOTECHNOLOGY
SUBJECT CODE- BP605T

Immunoblotting
TECHNIQUES
Chamanpreet Kaur
Assistant Professor
Pharmaceutics
ASBASJSMCOP, BELA

2
OBJECTIVE OF COURSE
Genetic Engineering application in relation to
production of Pharmaceutical

LEARNING OUTCOME:
Students will learn about ELISA and blotting
techniques. They will also learn about microbial
transformation and mutation.
 ELISA
Enzyme-linked immunosorbent assay (ELISA): ELISA
stands for "enzyme-linked immunosorbent assay." This is
a rapid immunochemical test that involves an enzyme (a
protein that catalyzes a biochemical reaction). It also
involves an antibody or antigen (immunologic molecules).
ELISA tests are utilized to detect substances that have
antigenic properties, primarily proteins (as opposed to
small molecules and ions such as glucose and potassium).
Some of these include hormones, bacterial antigens and
antibodies
TYPES OF ELISA
 DEFINITION OF BLOTTING

• Visualization of specific DNA , RNA & protein

among many thousands of contaminating molecules
requires the convergence of number of techniques
which are collectively termed BLOT transfer .
TYPES OF BLOTTING TECHNIQUES
w

Blotting
Blotting technique
technique

Southern Blot Northern Blot Western blot

It is used to detect It is used to detect It is used to detect
DNA. RNA. protein

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 IMMUNOBLOTTING
• Viral antigens are detected with a polyclonal or a MAb
onto nitrocellulose paper.

• After incubation, the protein bands (immune complexes)

are visualized with peroxidase-conjugated protein and a
colour reagent.

• A colour develops in the bands where antibody binds to

the antigen.

• Immunoblotting assay mixture of this two technique.

 WESTERN BLOTTING
• Western blotting is based on the principles of immunochromatography where proteins
were separated into poly acrylamide gel according to the isoelectric point and molecular
weight.

• A technique for detecting specific proteins separated by electrophoresis by use of labeled

antibodies.

• Immunoblotting is performed chiefly in diagnostic laboratories to identify the desirable

protein antigens in complex mixtures.
• An improved immunoblot method Zestern analysis, is able to address this issue without
the electrophoresis step, thus significantly improving the efficiency of protein analysis.

• Other related techniques include dot blot analysis, zestern analysis,

immunohistochemistry where antibodies are used to detect proteins in tissues and cells
by immunostaining and enzyme-linked immunosorbent assay (ELISA).
 WESTERN BLOT

Western Blot
• Lane1: Positive Control
• Lane 2: Negative Control
• Sample A: Negative
• Sample B: Indeterminate
• Sample C: Positive
 CONTENTS

• Tissue preparation
• Gel electrophoresis
• Transfer
• Blocking
• Detection
• Analysis
• Applications
 TISSUE PREPARATION
 Samples may be taken from whole tissue, from cell culture, bacteria, virus or
environmental samples.

 In most cases, samples are solid tissues.

 First broken down mechanically using a blender (for larger sample volumes), using a
homogenizer (smaller volumes), or by sonication.

 Cells may also be broken open by one of the above mechanical methods.
 A combination of biochemical and mechanical techniques, including various types of
filtration and centrifugation.
 To encourage lysis of cells and to solubilize proteins, may be employed : detergents,
salts, and buffers

 To prevent the digestion of the sample by its own enzymes -Anti Protease and
phosphatase

 To avoid protein denaturing-Tissue preparation is often done at cold temperatures

 GEL ELECTROPHORESIS
The proteins of the sample are separated using gel electrophoresis.
Separation of proteins may be by isoelectric point molecular weight,
electric charge, or a combination of these factors. Commercially SDS-
PAGE gel electrophoresis for protiens.

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 POLYACRYLAMIDE GEL
• Polymerized gel:
1. Resolving gels made in 6%, 10%,
12%, 18%.
2. Stacking Gel up to 5% was added
to gel and then the wells are
created.
• The percentage chosen depends on
the size of the protein that one
wishes to identify or probe in the
sample.
• The smaller it is the bigger the
percentage.
 SDS-PAGE (POLYACRYLAMIDE GEL
ELECTROPHORESIS)
• SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is
a technique widely used in biochemistry, forensics, genetics and molecular
biology
– to separate proteins according to their electrophoretic mobility.
– to separate proteins according to their size, and no other physical feature.

• SDS (the detergent soap) breaks up hydrophobic areas and coats proteins
with negative charges thus overwhelming positive charges in the protein.
• Therefore, if a cell is incubated with SDS, the membranes will be
dissolved, all the proteins will be solubilized by the detergent and all the
proteins will be covered with many negative charges.
 SDS PAGE
• The Pink Strands are the denatured Proteins covered in the
negatively charged SDS .

• See varying size they Are traveling to the positive since they have
negative charge.
• These strands go throught the tunnel and are seperated by their
size.
 PAGE
• If the proteins are denatured and put into an electric field (only), they
will all move towards the positive pole at the same rate, with no
separation by size.

• However, if the proteins are put into an environment that will allow
different sized proteins to move at different rates.

• The environment is polyacrylamide.

• the entire process is called polyacrylamide gel electrophoresis
(PAGE).

• Small molecules move through the polyacrylamide forest faster than

big molecules, big molecules stays near the well.
 TRANSFER
• In order to make the proteins accessible to antibody
detection they are moved from within the gel onto a
membrane made of nitrocellulose or polyvinylidene
difluoride (PVDF) . 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.
• Another method for transferring the proteins is called
electrobloting and uses an electric current to pull
proteins from the gel into membrane.
 Both varieties of membrane are chosen for their non-specific
protein binding properties (i.e. binds all proteins equally well).
Protein binding is based upon hydrophobic interactions, as
well as charged interactions between the membrane and
protein.

 The uniformity and overall effectiveness of transfer of protein

from the gel to the membrane can be checked by staining the
membrane with coomassie or ponceau S dyes.

 Nitrocellulose membranes are cheaper than PVDF, but are far

more fragile and do not stand up well to repeated probings.
 BLOTTING
• Blotting used to transfer the
samples from the gel on to a
membrane such as a nylon
membrane or nitrocellulose
membrane.

• Analyzed through probing

with nucleic acid probes or
antibody probes.
 BLOCKING
• Steps must be taken to prevent interactions between the membrane and
the antibody used for detection of the target protein (since the antibody is
a protein itself).
• Blocking of non-specific binding is achieved by placing the membrane
in a dilute solution of protein.

• Typically Bovin Serum Albumin (BSA) or non-fat dry milk (both are
inexpensive), with a minute percentage of detergent such as Tween20.
* The protein in the dilute solution attaches to the membrane in all
places where the target proteins have not attached.
* This reduces "noise" in the final product of the Western blot,
leading to clearer results, and eliminates false positives.
 DETECTION
• The membrane is "probed" for the protein of interest with a modified antibody
which is linked to a reporter enzyme, which when exposed to an appropriate
substrate drives a colourimetric reaction and produces a colour.

• - Two step
• Primary antibody {Antibodies are generated when a host species or immune cell
culture is exposed to the protein of interest (or a part thereof) }.
• A dilute solution of primary antibody (generally between 0.5 and 5
micrograms/mL) is incubated with the membrane under gentle agitation for
anywhere from 30 minutes to overnight at different temperatures.

• The solution is composed of buffered saline solution with a small percentage of

detergent, and sometimes with powdered milk or BSA.
• Secondary antibody {After rinsing the membrane to remove unbound primary antibody, the membrane
is exposed to another antibody, directed at a species-specific portion of the primary antibody }.

• The secondary antibody is usually linked to biotin or to a reporter enzyms such as alkalin phosphatase
or horseradish peroxidase. This means that several secondary antibodies will bind to one primary
antibody and enhance the signal.

• Most commonly, a horseradish peroxidase-linked secondary is used to cleave a chemiluminescent

agent, and the reaction product produces luminescence in proportion to the amount of protein.

• A cheaper but less sensitive approach utilizes a 4-chloronaphthol stain with 1% horseradish
peroxidase; reaction of peroxide radicals with 4-chloronaphthol produces a dark brown stain that can
be photographed without using specialized photographic film.

 ONE STEP
• Historically, the probing process was performed in two steps because of the relative ease of producing
primary and secondary antibodies in separate processes.

• One-step probing systems that would allow the process to occur faster and with less consumables.
• This requires a probe antibody which both recognizes the protein of interest and contains a detectable
label, probes which are often available for known proteins tags.
 ANALYSIS
• In practical terms, not all Westerns reveal protein only at one band in a membrane.
• Size approximations are taken by comparing the stained bands to that of the marker or
ladder loaded during electrophoresis.
• The process is repeated for a structural protein, such as actin or tubulin, that should not
change between samples.
• This practice ensures correction for the amount of total protein on the membrane in
case of errors or incomplete transfers.

1. COLORIMETRIC DETECTION :
• This method depends on incubation of the Western blot with a substrate that reacts with
the reporter enzyme (such as peroxidase) that is bound to the secondary antibody.
• This converts the soluble dye into an insoluble form of a different color that
precipitates next to the enzyme and thereby stains the membrane.

• Protein levels are evaluated through densitometry or spectrophotometry.

3. RADIOACTIVE DETECTION
• Radioactive labels do not require enzyme substrates, but rather allow the
placement of medical X-ray film directly against the western blot which
develops as it is exposed to the label and creates dark regions which correspond
to the protein bands of interest.

• Very expensive, health and safety risks are high.

4. FLUORESCENT DETECTION
• The fluorescently labeled probe is excited by light and the emission of the
excitation is then detected by a photosensor such as CCD camera.

• Allows further data analysis such as molecular weight analysis and a

quantitative western blot analysis.
• The most sensitive detection methods for blotting analysis.
 APPLICATIONS
• The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human
serum sample. Proteins from known HIV-infected cells are separated and blotted on a
membrane as above. Then, the serum to be tested is applied in the primary antibody
incubation step; free antibody is washed away, and a secondary anti-human antibody linked
to an enzyme signal is added. The stained bands then indicate the proteins to which the
patient's serum contains antibody.

• A western blot is also used as the definitive test for Bovine spongiform
encephalopathy (BSE, commonly referred to as 'mad cow disease').

• Some forms of Lyme disease testing employ western blotting.

• Western blot can also be used as a confirmatory test for Hepatitis B infection.

• In veterinary medicine, western blot is sometimes used to confirm FIV+ status in cats.
 WHEN SHOULD WB BE USED
• Western blot assay should not be used as a screening test.

• Wb should be viewed as a supplemental test which can be used to confirm

positive results obtained from enzyme immuno assay (EIA).

• However:

– Specificity is less than that of EIA.

– A significant number of indeterminate blots are seen in low risk populations.

DISADVANTAGES
• If a protein is degraded quickly, Western blotting won't detect it well.
• This test takes longer that other existing tests.
• It might also be more costly.
Mutation
 Introduction
• Sudden heritable change in genetic material or character of an organism is
known as mutation

• Individuals showing these changes are known as mutants

• An individual showing an altered phenotype due to mutation are known as

variant

• Factor or agents causing mutation are known as mutagens

• Mutation which causes changes in base sequence of a gene are known as

gene mutation or point mutation
 History
• English farmer Seth Wright recorded case of mutation first time in 1791 in
male lamb with unusual short legs

• The term mutation is coined by Hugo de Vries in 1900 by his observation

in Oenothera

• Systematic study of mutation was started in 1910 when Morgan

genetically analyzed white eye mutant of Drosophila

• H. J. Muller induced mutation in Drosophila by using X- rays in 1927 ; he

was awarded with Nobel prize in 1946
 Characteristics of Mutation
• Generally mutant alleles are recessive to their wild type or normal alleles

• Most mutations have harmful effect, but some mutations are beneficial

• Spontaneous mutations occurs at very low rate

• Some genes shows high rate of mutation such genes are called as mutable
gene

• Highly mutable sites within a gene are known as hot spots.

• Mutation can occur in any tissue/cell (somatic or germinal) of an organism

 Classification of mutation
• Based on the survival of an individual

1. Lethal mutation – when mutation causes death of all individuals undergoing

mutation are known as lethal

2. Sub lethal mutation - causes death of 90% individuals

3. Sub vital mutation– such mutation kills less than 90% individuals

4. Vital mutation -when mutation don’t affect the survival of an individual are
known as vital

5. Supervital mutation – This kind of mutation enhances the survival of

individual
• Based on causes of mutation
1. Spontaneous mutation-Spontaneous mutation occurs naturally without
any cause. The rate of spontaneous mutation is very slow eg- Methylation
followed by deamination of cytosine. Rate of spontaneous mutation is
higher in eukaryotes than prokaryotes.
Eg. UV light of sunlight causing mutation in bacteria

2. Induced Mutation-Mutations produced due to treatment with either a

chemical or physical agent are called induced mutation .The agents capable
of inducing such mutations are known as mutagen.use of induced mutation
for crop improvement program is known as mutation breeding.
Eg. X- rays causing mutation in cereals
• Based on tissue of origin

1. Somatic mutation-
A mutation occurring in somatic cell is called somatic mutation.
In asexually reproducing species somatic mutations transmits from one
progeny to the next progeny

2. Germinal Mutation-
When mutation occur in gametic cells or reproductive cells are known as
germinal mutation.
In sexually reproductive species only germinal mutation are transmitted to the
next generation
• Based on direction of mutation

1.Forward mutation- When mutation occurs from the

normal/wild type allele to mutant allele are known as forward
mutation

2.Reverse mutation- When mutation occurs in reverse direction

that is from mutant allele to the normal/wild type allele are
known as reverse mutation
• Type of trait affected

1. Visible mutation- Those mutation which affects on

phenotypic character and can be detected by normal
observation are known as visible mutation

2. Biochemical mutation- mutation which affect the production

of biochemicals and which does not not show any
phenotypic character are known as biochemical mutation
 Chromosome Mutations

• May Involve:
– Changingthe
structure

loss or gain
 Chromosome Mutations
• Five types exist:
–Deletion
–Inversion
–Translocation
–Nondisjunction
–Duplication
 Deletion
• Due to breakage
• A piece of a
chromosome is lost
 Inversion

• Chromosome segment
breaks off
• Segment flips around
backwards
• Segment reattaches
 Duplication
• Occurs when a gene sequence is repeated
 Translocation
• Involves two chromosomes that aren’t
homologous
• Part of one chromosome is transferred to
another chromosomes
Translocation
 Nondisjunction
• Failure of chromosomes to
separate during meiosis
• Causes gamete to have too many
or too few chromosomes
• Disorders:
– Down Syndrome –
– Turner Syndrome –
– Klinefelter’s Syndrome –
 Types of Gene Mutations
• Include:
–Point Mutations
–Substitutions
–Insertions
–Deletions
–Frameshift
 Point Mutation

• Change of a single
nucleotide
• Includes the deletion,
insertion, or substitution
of ONE nucleotide in a
gene
 Point Mutation

• Sickle Cell disease is

the result of one
nucleotide
substitution
• Occurs in the
hemoglobin gene
 Frameshift Mutation

• Inserting or deleting one or more

nucleotides
• Changes the “reading frame” like
changing a sentence
• Proteins built incorrectly
 MICROBIAL
BIOTRANSFORMATION
 BIOTRANSFORMATION
Biotransformations are structural modifications in a chemical
compound by organisms /enzyme systems that lead to the
formation of molecules with relatively greater polarity.This
mechanism has been developed by microbes to acclimatize to
environmental changes and it is useful in a wide range of
biotechnological processess. The most significant aspect of
biotransformation is that it maintains the original carbon
skeleton after obtaining the products.
TYPES OF BIOTRANSFORMATION

Biotransformation is of two types:

1.Enzymatic: Microsomal biotransformation is caused
by enzymes present within the lipophilic membranes of
smooth endoplasmic reticulum .
2.Nonenzymatic: Non-Microsomal Biotransformation
involves the enzymes which are present within the
mitochondria.
Microbial cells are ideal choice for biotransformation
due to certain reasons like:
I. Surface-volume ratio: Microbial biotransformation has high
surface-volume ratio.
II. Growth Rate: Higher growth rate of microbial cells reduces the
time of biomass transformation.
III. Metabolism Rate: Higher rate of the metabolism in microbes leads
to efficient transformation of substrate.
IV. Sterility: It is easier to maintain sterile conditions when microbes
are used
 APPLICATION OF MICROBIAL
BIOTRANSFORMATION

•Transformation of steroids and sterols.

•Transformation of Pollutants.
•Transformation of Non-Steroid Compounds.
•Transformation of Antibiotics.
•Transformation of Pesticides.
•Petroleum Biotransformation.
GENETIC ORGANIZATION
OF EUKARYOTES AND
PROKAROYOTES
 EUKARYOTES AND
PROKAROYOTES
Prokaryotes are organisms made up of
cells that lack a cell nucleus or any
membrane-encased organelles.
Eukaryotes are organisms made up of
cells that possess a membrane-bound
nucleus that holds genetic material as well
as membrane-bound organelles.
DIFFERENCE BETWEEN EUKARYOTES
AND PROKAROYOTES
TRANSFORMATION: Transformation occurs naturally in some species of bacteria,
and can also be done artificially. ... Introduction of foreign DNA into eukaryote cells is usually
called "transfection".
TRANSDUCTION: Transduction is the process by which foreign DNA is
introduced into a bacterial cell by a virus or viral vector. An example is the viral transfer of
DNA from one bacterium to another and hence an example of horizontal gene transfer.
CONJUGATION: Bacterial conjugation is the transfer of genetic material
between bacterial cells by direct cell-to-cell contact or by a bridge-like
connection between two cells.This takes place through a pilus.
PLASMIDS: A plasmid is a small, circular, double-stranded DNA molecule that is
distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they
also occur in some eukaryotes. Often, the genes carried in plasmids provide bacteria
with genetic advantages, such as antibiotic resistance