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Advance Techniques in Protein Estimation
SINHGAD INSTITUTE OF PHARMACY, NARHE
Presented by
Guide
Mr. Gogawale Vinayak G.M. Pharm. (Semester-III) (Department of Pharmacology)
Dr. U. M. Aswar(Asst. Professor Pharmacology)
CONTENTS
Introduction
Techniques for protein estimation
Immuno histochemistry
PCR
Blotting techniques
SDS Page
Spectroscopy
03/05/2023 2
Techniques for protein estimation
• Immuno histochemistry
• PCR
• Blotting techniques
• SDS Page
• Spectroscopy
03/05/2023 4
Immuno histochemistry
• Immunohistochemistry is the technique in which
combination of histological, immunological and
biochemical techniques for the identification of
specific tissue components.
• Antigen/antibody reaction labeled with a visible label.
• Possible to identify any cell component within cell or
tissue.
03/05/2023 5
Cont….
• Requirements for
immuno-histochemistry :
• Antibody
• Antigen Retrieval
• Blocking
• Methods for IHC:
• Direct method
• Indirect method
• Immunoenzyme
• Fluorescence
• Multiple labeling
03/05/2023 6
03/05/2023 7
• Antibodies:• Immune cells, or B cells, produce antibodies against
targeted proteins.
• They have two types Monoclonal and polyclonal
antibodies.
• a monoclonal antibody preparation contains a single
antibody with specificity to one epitope on the antigen
molecule.
• Polyclonal antibodies are different B cell produced
antibodies against different sites on the same antigen.
• Antigen Retrieval: Routine histology procedures
masks the antigens, usually by formalin cross-linking,
or even destroys some antigen epitopes.
03/05/2023 9
Blocking
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In principle, any protein that does not specifically bind
to your epitope of interest, or to the antibodies in your
immunohistochemistry assay, can be used to block.
The samples are incubated with a buffer that blocks the
reactive sites to which the primary or secondary
antibodies may otherwise bind. Common blocking
buffers include normal serum or BSA.
• It is method for amplifying particular segments of
DNA, distinct from cloning and propagation within
the host cell.
• The procedure is carried out entirely biochemically,
that is, in vitro.
• PCR was invented by Kary Mullis in 1983. He
shared the Nobel Prize in chemistry with Michael
Smith in 1993.
Polymerase Chain Reaction
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PRINCIPLE
• PCR uses the enzyme DNA polymerase that directs
the synthesis of DNA from deoxynucleotide substrates
on a single-stranded DNA template.
• DNA polymerase adds nucleotides to the 3` end of a
custom-designed oligonucleotide when it is annealed
to a longer template DNA.
• DNA polymerase can use the oligonucleotide as a
primer and elongate its 3` end to generate an extended
region of double stranded DNA.03/05/2023 14
PCR Steps:
• Denaturation step (90–95 °C)
This breaks the weak hydrogen bonds that hold DNA
strands together in a helix
It allowing the strands to separate creating single stranded
DNA.
• Annealing step (50–65 °C)
This allows the primers to bind (anneal) to their
complementary sequence in the template DNA.
Cont….
03/05/2023 15
• Extension/Elongation step (up to 72° C)
DNA polymerase extends the primers, adding nucleotides onto
the primer in a sequential manner, using the target DNA as a
template.
With one cycle, a single segment of double-stranded DNA
template is amplified into two separate pieces of double-
stranded DNA. These two pieces are then available for
amplification in the next cycle. As the cycles are repeated,
more and more copies are generated and the number of copies
of the template is increased exponentially.
Cont….
03/05/2023 16
Blotting Techniques
Southern blotting
•Used for transferring DNA•Hybridization•Nitrocellulose filter membrane
Northern blotting
•Used for transferring RNA •Hybridization•Amonobenzyloxy methyl filter paper
Western blotting
• Used for transferring Proteins
• Nitrocellulose filter membrane
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Southern Blotting• Southern blot is a method used to check for the
presence of a DNA sequence in a DNA sample.
• Hybridization of proteins is the main process.
• The principle of hybridization analysis is that a
single-stranded DNA or RNA molecule of defined
sequence (the probe) can base-pair to a second DNA
or RNA molecule that contains a complementary
sequence (the target), with the stability of the hybrid
depending on the extent of base pairing that occurs. 03/05/2023 19
Northern Blot
• The northern blot technique is used to study gene
expression by detection of RNA (or isolated mRNA)
in a sample.
• With northern blotting it is possible to observe
cellular control over structure and function by
determining the particular gene expression levels
during differentiation, morphogenesis, as well as
abnormal or diseased conditions.03/05/2023 21
Western Blotting
• Western blotting is an Immunoblotting technique
which rely on the specificity of binding between a
molecule of interest and a probe to allow detection of
the molecule of interest in a mixture of many other
similar molecules.
• It is dependent on the quality of antibody use for probe
to protein of interest, and how specific it is for this
protein03/05/2023 23
Steps involved in western blotting
• Tissue preparation
• Gel electrophoresis
• SDS PAGE
• Transfer
• Blocking
• Detection
• Analysis03/05/2023 24
Cont….
SDS PAGE
• Sodium dodecyl sulfate poly acrylamide gel
electrophoresis.
• Poly acrylamide gel as a support medium and sodium
dodecyl sulphate (SDS) to denature the proteins. The
method is called sodium dodecyl sulphate poly
acrylamide gel electrophoresis (SDS-PAGE).
• The purpose of SDSPAGE is to separate proteins
according to their size.03/05/2023 25
• SDS is an anionic detergent, meaning that when
dissolved its molecules have a net negative charge
within a wide pH range. A polypeptide chain binds
amounts of SDS in proportion to its relative molecular
mass. The negative charges on SDS destroy most of
the complex structure of proteins, and are strongly
attracted toward an anode (positively-charged
electrode) in an electric field.
03/05/2023 26
Cont….
• The final separation of proteins is dependent almost
entirely on the differences in relative molecular mass
of polypeptides.
• If proteins of known mass are run simultaneously
with the unknowns, the relationship between Rf
(distance travelled by proteins) and mass can be
plotted, and the masses of unknown proteins
estimated.
03/05/2023 27
Cont….
Transferring• 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 electro
blotting and uses an electric current to pull proteins from the
gel into the PVDF or nitrocellulose membrane.03/05/2023 28
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.
• Blocking of non-specific binding is achieved by placing the
membrane in a dilute solution of protein - typically Bovine serum
albumin with a minute percentage of detergent such as Tween 20.
The protein in the dilute solution attaches to the membrane in all
places where the target proteins have not attached.
• Thus, when the antibody is added, there is no space on the
membrane for it to attach other than on the binding sites of the
specific target protein.03/05/2023 29
Detection
03/05/2023 30
• During the detection process, 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
colorimetric reaction and produces a color.
MALDI- TOFMS• Matrix Assisted Laser Desorption/Ionization- Time of
Flight Mass Spectrometry.
• The sample for MALDI is uniformly mixed in a large
quantity of matrix.
• The matrix absorbs the ultraviolet light (nitrogen laser
light, wavelength 337 nm) and converts it to heat energy.
• A small part of the matrix heats rapidly (in several nano
seconds) and is vaporized, together with the sample.
03/05/2023 32
• MALDI-Tof-MS for identification of protein disease
markers
• It has been developed for use in clinical chemistry as
a primary investigative tool to characterize cancer,
Alzheimer, arthritis, and allergy protein markers of
disease or susceptibility to disease.
APPLICATIONS
03/05/2023 34
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“Principles and applications of polymerase chain reaction in medical
Diagnostic fields: a review”, Brazilian Journal of Microbiology (2009)
40:1-11.03/05/2023 36
• Marvin L. F., Roberts M. A., Flay L. B., “Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry in clinical
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• Liu Y. S., Huang Z. W., Wang L., Liu X. X., “Sitagliptin
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of Pharmacological Sciences (2015), 127, 260-274.
03/05/2023 37
Cont….