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

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Introduction

• Proteins are the .

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Techniques for protein estimation

• Immuno histochemistry

• PCR

• Blotting techniques

• SDS Page

• Spectroscopy

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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.

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Cont….

• Requirements for

immuno-histochemistry :

• Antibody

• Antigen Retrieval

• Blocking

• Methods for IHC:

• Direct method

• Indirect method

• Immunoenzyme

• Fluorescence

• Multiple labeling

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• 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.

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Cont….Method for Monoclonal antibodies

• Antigen Retrieval: Routine histology procedures

masks the antigens, usually by formalin cross-linking,

or even destroys some antigen epitopes.

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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.

Methods for IHC

Direct method Indirect method

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• 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….

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• 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….

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Cont….Application of PCR

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

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Cont….Procedure for Southern Blot

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

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Cont….Procedure for blot

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.

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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.

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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

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• 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.

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Cont….Application of western blot

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.

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Procedure

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• 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

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Cont….

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“Principles and applications of polymerase chain reaction in medical

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Cont….