This document provides an overview of several molecular pharmacology techniques: 1. Western blotting is used to detect specific proteins and involves separating proteins by size, transferring them to a membrane, and using antibodies to visualize target proteins. 2. Immunostaining uses antibodies to detect specific proteins in tissue samples and can be used for diagnostic purposes. 3. RT-PCR is used to analyze gene expression by converting mRNA to cDNA and then amplifying specific sequences using PCR for detection. 4. Cloning techniques such as DNA cloning are used to generate identical copies of DNA fragments or genes using vectors like bacterial plasmids.

1. Techniques for the study of
Molecular pharmacology
Prof. Mrs. Pradnya Jagtap
Ms. Bhagyashri R. Vyapari
Ms.Prachali Chavan
Pharmacology
PDEA’s S.G.R.S. College of Pharmacy, Saswad, Pune.

2. Contents
• Western blotting
• Immunostaining
• RT- PCR
• Cloning
• RIA

3. Western blotting
• Blotting:
Blots are techniques for transferring DNA, RNA &
proteins onto a carrier (for e. g. , a nitrocellulose or
PVDF or nylon membrane) so they can be separated,
and often follows the use of a gel electrophoresis.
The southern blot is used for transferring DNA, the
northern blot is used for RNA and the western blot
for PROTEINS

4. Types of blotting techniques
BLOTTING
TECHNIQUES
Southern blotting
Used to detect
DNA
Northern blotting
Used to detect
RNA
Western blotting
Used to detect
Proteins

5. Western blotting
• Western blotting is widely used analytical technique in
molecular biology to detect specific protein in a sample
of tissue homogenate or extract.
• The technique uses three elements to accomplish this
task:
1) Separation by size
2) Transfer to a solid support
3) Marking target protein using a proper primary &
secondary antibody to visualize.
• In this technique a mixture of proteins is separated
based on molecular weight, and thus by type, through
gel electrophoresis.

6. Flow
chart of
western
blotting
procedure

7. Sample preparation
Detergent lysis
For tissue culture
Ultrasonocation
For cell suspension
Mechanical
homogenisation
For plant and
animal tissue
Enzymatic digestion
For bacterial, yeast
and fungal cells
Gel electrophoresis
• Electrophoresis is commonly used
method for separating proteins on
the basis of size, shape or charge
•In gel electrophoresis, protein of
sample extract are separated according
to molecular weight.

9. Protein 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.
Fig. transfer of protein on
nitrocellulose membrane

10. Protein staining
• 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, silver stain, or deep purple.
• After staining, a permanent record may be made by
imaging the gel with suitable instrument.

12. Blocking
• For meaningful results, the antibodies must bind only
to the protein of interest and not to the membrane.
• Non-specific binding (NSB) 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.

13. Blocking agents
• 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

14. 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 a membrane.
• The specificity of antigen- antibody binding 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 the primary antibody.

15. • The secondary antibody is conjugated to an enzyme
that is used to indicate the location of the protein.
• Secondary antibodies can be a monoclonal or
polyclonal antibodies.
• The secondary antibodies not only serves as a carrier
of the label, but it is also helps to amplify the emitted
signals.
• The signal emitted by the labelled secondary
antibody is then measured and is proportional to the
quantity of protein of interest present on the
membrane.

16. Washing
• 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.

17. Protein detection
• After the unbound probes are washed away, the
western blotting is now ready for detection of the
probes that are labeled and bound to the protein of
interest.
• Enzymes such as alkaline phosphatase (AP), &
Horse- radish peroxidase (HRP) are widely used in
detection of proteins.

18. • There are four methods of detection and they are as
follows:
1. Chromogenic detection
2. Chemiluminescence detection
3. Fluorescent detection
4. Radioactive detection
Analysis and imaging
• This is the last and major step
of the western blotting technique.
Fig. CCD used in western blotting

19. • Detection of signals, using either – ray film, scanners
or a CCD, results in one or more visible protein
bands on the membrane image.
• The molecular weight of the protein can be estimated
by comparison with marker proteins and the amount
of protein can be determined as 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.

20. Applications
• Analysis of IgG fractions purified from human
plasma.
• Diagnosis of HIV by ELISA involves the western
blotting techniques.
• It is also used to detect some forms of Lyme disease.
• It is used in definitive test for BSE, which is
commonly known as mad cow disease.
• Confirmatory test for hepatitis-B involves western
blotting technique.

21. • 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.
Limitations of western blotting
• Very delicate and time consuming process. A minute
imbalance any level of the procedure can skew the
results of the entire process.
• Incorrect labeling of the protein can happen due to
the reaction of secondary antibody.

22. • Cause erroneous in bands or no bands due to
insufficient transfer.
• Well trained technicians are required for this
technique.
• Primary antibody availability is crucial.
• It is just a semi- quantitative at best. Only an approx
estimation & not a precise measurement of molecular
weight of the protein is possible.

23. Immunostaining
• Immunostaining is a general term
in biochemistry that applies to any
use of an antibody- based method to
detect a specific protein in a sample.
• The term immunostaining was originally used to refer
to the immunohistochemical staining of tissue
sections, as first described by Albert Coons in 1941.
• The key to immunohistochemistry is the specific
antibody- mediated detection of target antigen,
known as immunostaining or immunodetection.

24. Immunostaining method
According to different biotins conjugated with
antibodies, IHC staining methods can be classified as
• Immunofluorescence,
• Immunoenzymological staining
• Immunocolloidal gold technique
According to different kinds of procedures,
immunohistochemistry staining can be divided into
subtypes of
• Direct staining (one- step staining)

25. • Indirect staining(two step, three step or multi step
staining)
• PAP staining method
Different biotins conjugated with
antibodies
• Immunofluorescence:
It is the first immunohistochemical
staining method with antigen
antibody binding reaction, antigens
are visualized by fluorescence dyes conjugated with
antibodies and that is localized under fluorescence
microscope.

26. Immunoenzymological staining:
• In here enzyme- labeled antibodies
are used to bind with specific antigens
in tissues samples or cultured cells
and localized by light microscope.
Micro colloidal gold technique:
• It is a kind of technique that uses
colloidal gold as a marker.
Immunocolloidal gold technique is
suitable for single or multi- label
detection under immune- electron microscope, and
light microscope.

27. Different kinds of procedures
• Direct staining:
Incubate the sections with mixture
of two primary antibodies which are
respectively conjugated with two
fluorescence dyes (e.g. FITC and
TRITC) or, successively incubate
sections with two primary antibodies.
• Indirect staining:
In here the primary antibodies are without
fluorescence dyes. Incubate sections with one kind of
primary antibody and corresponding secondary
antibody, then the other.

28. PAP Staining method (peroxidase anti-
peroxidase method):
• Incubate sections with
corresponding secondary
antibodies which are
conjugated with two different
enzymes (e. g. HRP, AKP), or
anti- HRP (PAP complex),
anti- AKP (APAAP complex).

30. Immunohistochemistry staining tips
• Fresh fixed tissue
• Sufficient dehydration of tissues
• An intact, uniform and smooth sectioning
• Binder materials
• Sufficient deparaffinization
• A thorough inhibition of endogenous peroxidase

31. Applications
• Clinically, IHC is used in histopathology for the
diagnosis of types of cancers based on molecular
markers.
• In laboratory science, immunostaining can be used
for a variety of applications based on investigating
the presence or absence of a protein, its tissue
distribution, its sub-cellular localization, and of
changes in protein expression or degradation.

32. RT- PCR (Reverse transcriptase
polymerase chain reaction)
Fig. PCR

33. PCR (polymerase chain reaction):
It is technique used in molecular biology to amplify a
single copy or a few copies of a segment of DNA
across several orders of magnitude, generating
thousands to millions of copies of a particular DNA
sequence.
Features:
• Sensitive, selective and rapid
• Allows DNA in a single cell/ hair follicle/
spermatozoa to be amplified
• 20 cycles- amplification of 10 raised to 6 times
30 cycles- amplification of 10 raised to 9 times

34. Steps:

35. 1. Strand separation (Denaturation):
DNA strands are separated by heating at 95ºC for
15 sec- 2 min.
2. Primer annealing:
solution is cooled at 55ºC and primers are added
primers hybridized with DNA produced in step 1
3. Polymerization:
synthesis of new DNA strands by addition of
dNTPs
enzyme: Taq DNA polymerase
(Heat stable polymerase derived from Thermus
Aquaticus)

36. Reaction is carried out at 72ºC for 30- 60 sec
Steps 1, 2, 3 are repeated for 20- 30 cycles
(thermocycler/ tempcycler)

37. PCR: Types
• Reverse transcriptase PCR (RTPCR)
Detection of mRNA
• Nested PCR
two sets of primers are used
reduce the contamination of PCR products
• Real time PCR
used to quantify the virus load
• Quantitative PCR
Used to measure the amount of DNA

38. • RACE- PCR (Rapid Amplification of cDNA ends)
used to amplify ends of mRNA
• Multiplex- PCR
multiple primers are used in single PCR reaction
Equipments required:
• PCR machine
• Biosafety cabinet
• 96 well plates
• Pipettes
• Microcentrifuge tubes
• Table top centrifuge
• Tips with tips boxes
• Cool box

40. Advantages:
• Most accurate and feasible technique to determine the
amount & concentration of products.
• Rapid cycling (30 min. to 2 hours)
• Specific and sensitive
• Not much expensive
Disadvantages:
• Pollution/ contamination
• Poor precision
• Hard to get quantitative data

41. PCR applications:
• Diagnosis of infectious (HIV/ TB)/ genetic diseases
(cystic fibrosis)
• Cancer detection
• Forensic applications
• Disputes of parenthood
• Prenatal diagnosis
• To study evolution
• To establish precise tissue type for transplant

42. RT- PCR
• Reverse transcriptase is a common name for an
enzyme that functions as a RNA- dependent DNA
polymerase.
• They are encoded by retroviruses, where they copy
the viral RNA genome into DNA prior to its
investigation into host cells.
• In the laboratory, it is used for analyzing gene
expression, i. e. convert mRNA to cDNA by reverse
transcription.

43. • Reverse transcriptase have two activities:
1. DNA polymerase activity
2. RNAse H activity
• All retrovirus have a reverse transcriptase, but the
enzymes that are available commercially are
derived from one of two retroviruses, either by
purification from the virus or expression in E. coli:
a. Moloney murine leukemia virus
b. Avian myeloblastosis virus
The technique consist of two parts:
1. The synthesis of cDNA (complimentary DNA)
from RNA by reverse transcription (RT)

44. 2. The amplification of a specific cDNA by PCR
RNA- directed DNA polymerase (rTh) yields ds
cDNA

45. Advantages:
• Automated, fast, reliable (reproducible) results
• Contained less chances of contamination
• High output
• Sensitive
• Broad uses
• Defined, easy to follow protocols
Disadvantages:
• Need for equipment
• Taq polymerase is expensive
• Contamination
• False reactions
• Internal control

46. • Cross- reaction
• Enrichment steps in (contaminated) samples
• Capacity building needed
• Unspecific amplification
Applications:
• Genome mapping and gene function determination
• Biodiversity studies (e. g. evolution studies)
• Diagnostics (prenatal testing of genetic diseases,
early detection of cancer, viral infections)
• Detection of drug resistance genes
• Forensics (DNA fingerprinting)

47. • From the Greek word- klon, a twig
• Cloning is the production of genetically identical
individuals that have identical nuclear DNA.
Cloning technologies:
1. Recombinant DNA technology
• DNA cloning
• Molecular cloning
• Gene cloning
2. Reproductive cloning
3. Therapeutic cloning
• Embryo cloning
Cloning

48. • The technique of generating identical copies of short
stretches of DNA is referred to as molecular cloning.
• DNA cloning refers to replication of a single DNA
molecule to create a large number of identical DNA
molecules.
• DNA cloning are of two types:
1. Cell based DNA cloning
2. Cell free DNA cloning
Cell based DNA cloning:
• DNA is extracted from blood
• Restriction enzymes,
e. g. EcoRI, HindIII, etc.,
cut the DNA into small pieces

49. • Different DNA pieces cut with the same enzyme can
join, or recombine.
Cloning vectors:

52. DNA cloning I

53. DNA cloning II
• Bacterial plasmids (small
circular DNA additional to
bacteria’s regular DNA) are
cut with the same restriction
enzyme
• A chunk of DNA can thus
be inserted into the plasmid
DNA to form a recombinant.

54. DNA cloning III
• The recombinant
plasmids are then
mixed with bacteria
which have been
treated to make them
competent, or capable
of taking in the plasmids.
• This insertion is called
transformation.

55. DNA cloning IV
• The plasmids have
naturally occurring
genes for antibiotic
resistance.
• Bacteria containing
plasmids with these
genes will grow on a
medium containing the
antibiotic- the others
die, so only transformed
bacteria survive.

56. DNA cloning V
• The transformed
bacterial cells form
colonies on the
medium
• Each cell in a given
colony has the same
plasmid (& the same
DNA)
• Cells in different
colonies have different
plasmids (& different
fragments)

57. Cell free DNA cloning
• In this method the PCR
technique is used to clone
the DNA.
DNA cloning Uses:
• Gene therapy
• Genetic engineering of organisms
• Genome sequencing

58. Reproductive cloning
• A technology used to generate an animal that has
same nuclear DNA as another currently or previously
existing animal
• Dolly
• Reproductive cloning done by somatic cell nuclear
transfer (SCNT)
Sources for SCNT:
1. Cell from individual
2. Cells grown in culture
3. Frozen tissue

59. SCNT:
• Starts with removal of polar body and chromosomes
from an oocyte (Enucleated oocyte)
• Donor cell then inserted into perivitelline space of
enucleated oocyte
• Oocyte and donor cell are fused and activated by an
electric pulse to begin cell division
• Developed embryos transferred to surrogate females
• Birth of an individual.

60. Therapeutic cloning
• Stem cells:
cells have ability to divide and give rise to both
specialized cells and more stem cells.
• Derived from:
- Adults
- Pre implantation embryos (embryonic stem cells)

61. Stem cells:
• Replacement cell to treat:-
- Heart disease
- Alzheimer’s
- Cancer
- Diabetes
- Parkinson’s disease
- Spinal cord injury

62. Applications of cloning
Agricultural applications:
• Herbicide resistance:
broadleaf plants have been
engineered to be resistant
to the herbicide
E. g. glyphosate
• Pest resistance:
insecticidal proteins have been transferred into crop
plants to make them pest resistant.
E. g. Bt toxin: from Bacillus thuringiensis

63. • Golden rice:
rice that has been genetically modified to produce
beta- carotene (provitamin A)
converted in the body to vitamin A
• Biopharming:
transgenic plants are used to produce pharmaceuticals
- Human serum albumin
- Recombinant subunit vaccines
Against Norwalk and rabies viruses
- Recombinant monoclonal antibodies
Against tooth decay causing bacteria

64. Transgenic mammals

65. Medical applications
• The insertion of genetic material into human cells for
the treatment of a disorder
• Recombinant DNA vaccines:-

66. • Gene therapy:-

67. Radio Immuno Assay
Immuno- assay:
• Immuno: it refers to the response that cause body to
generate antibodies.
• Assay: refers to test
• Immunoassay: is a test that use immuno-complexing
when antigen and antibodies are brought together.
Types of immunoassay:
1. Radio- immuno assay (RIA)
2. Enzyme immuno assay (ELISA)
3. Fluorescence polarization immuno assay (FPIA)

68. Principle
• RIA involves competitive binding of radio- labeled
antigen and unlabeled antigen to a high affinity
antibody.
• It involves separation of protein (from mixture) using
specificity of Ag- Ab and quantification using
radioactivity.
• The antigen is generally labeled with a gamma-
emitting isotope such as tritium (3 H) are also
routinely used as labels.
• This radiation is measured by beta or gamma
counters.

69. • Based on a competitive binding reaction between
- Fixed amount of a labeled analyte (Ag*)
- Variable amount of unlabeled sample analyte (Ag)
• For a limited amount of binding sites on a highly
specific antibody
Ab + Ag + Ag* Ag*- Ab + Ag- Ab + Ag* + Ag
Phase separation
Ag*- Ab + Ag- Ab Ag* + Ag
Bound fraction Free Fraction

70. Requirements of RIA
• Preparation and characterization of antigen
(ligand to be analysed)
• Radiolabelling of antigen
• Preparation of specific antibody
• Development of assay system
Preparation and radiolabelling of Ag:
Antigen preparation by:
• Molecule synthesis
• Natural source

71. • Radiolabelling (tagging procedure)
• 3H, 14C, 123I are used as radioactive tags
• Antigens are tagged to 3H, 14C, 123I.
(tagging should not affect the specificity and activity of
the antigen).
Preparation of specific antibody:
• Antigen injected intra dermally to rabbits or guinea
pigs – antibody production
• Antibodies recovered from the serum.
Development of assay system:
• Crucial step is separation of unbound antigens.
• Antibodies bind to the micro-titer well surface (solid
surface RIA).

72. • Antigens bound to the fixed antibodies remain stuck
to the inner surface.
• Decanting and washing the wells remove unbound
antigens.
• Other techniques of separations: centrifugation,
precipitation and electrophoresis.
Intensity of radioactivity is inversely proportional to
the concentration of antigen in test sample.
Assay procedure:

73. Unknown analyte concentration is determined from this curve.
Standard curve
Radioactivity measured in bound or free fraction
The bound antigens are separated from the unbound ones.
Known antigen will compete with the unknown patient antigen for sites on
the antibody
Mix known labeled antigen + patient sample + reagent antibody

75. Separation techniques
• Gel filtration chromatography
• Electrophoresis
• Charcoal dextran adsorption
• Adsorption on ion- exchange resin
Physical
methods
• Organic solvents such as ethanol,
PEG
• Salt such as ammonium sulphate
Chemical
methods

76. Pellet is formed at
an angle
Pellet is formed at
the bottom of the
test tube
3500- 4000
rpm
1200- 2500
rpm
Using a fixed-
angle- head
rotor
Using swing-
bucket- rotor
Two most vital equipments:
• Centrifuge
• Radioactive counters
Instrumentation
centrifuge

77. Radioactivity counters
Gamma counters
Used for counting
gamma- energy
emitting isotopes
125 I- iodine
isotope
Scintillation
counters
Used for counting
beta- energy emitting
isotopes
3H- tritium
14C- carbon
isotope

78. Advantages:
• Highly specific: immune reactions are specific
• High sensitivity: immune reactions are sensitive
• Detect few picograms (10 raised to -12 g) of antigen
in the tube.
Disadvantages:
• Radiation hazards
• Requires specially trained person
• Labs require special license to handle radioactive
material
• Requires special arrangements for
- requisition, handling, storage of radioactive material
- Radioactive waste disposal

79. Applications
• Screening donated blood
- Hepatitis C
- Hepatitis B
• Measuring hormone levels
- LH
- TSH, T3 & T4
- Hormones (e. g. anabolic steroids, HGH)
• Early cancer detection and diagnosis
• Measuring toxins in contaminated food

80. • Therapeutic drug monitoring:
- barbiturates, morphine, digoxin
• Detecting infections:
- Sexually transmitted agents like: HIV, syphilis &
Chlamydia
• Measuring rheumatoid factors & other auto
antibodies in autoimmune diseases.