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PROTEIN ANALYSIS

GOVERNMENT COLLEGE UNIVERSITY, LAHORE.


DEPARTMENT OF CHEMISTRY
AMINA INAM
22-BH-BT-2017
“ROLE OF PROTEIN ANALYSIS IN
BIOTECHNOLOGY”
SIR-SHAH HUSSAIN
7-06-2019

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PROTEIN ANALYSIS

TABLE OF CONTENTS:-
* ABSTRACT
* KEY WORDS
* INTRODUCTION
* PROTEIN AND IT’S STRUCTURE
* TECHNIQUES OF PROTEIN ANALYSIS
* PROTEIN SEPARATION
* SDS-PAGE
* ISO-ELECTRIC FOCUSING
*PROTEIN SEQUENCING
* EDMAN DEGRADATION
* CHROMIC METHODS
* MASS SPECTROSCOPY
* ELISA
* WESTERN BLOTTING
* GEL ELECTROPHORESIS
*REFERNCES

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Abstract:
Being one of the impressing groups of macromolecules, proteins and their
application are crucial to understand the molecular logic of living cells. Being
composed of amino acids despite their similar structure, proteins are surrounded by
several other molecules of different chemical structures creating an overcrowded
environment that makes them different in their functions. Unraveling the
importance of protein folding, protein–protein interaction is critical to understand
cellular processes and rational drug design. A major breakthrough in protein
purification technologies has greatly changed the landscape of protein study
leading to a deeper understanding of their role in disease biochemical pathway and
also in drug metabolism. Since the first protein sequencing in 1953 by Frederic
Sanger, proteomics, which is the large-scale study of proteins, became an essential
tool in understanding the protein structures and functions. Furthermore, the
application of physicochemical measures for protein content has lit up comparative
and functional biology of proteins. This chapter is intended to give a rudimentary
understanding of the methods and technologies behind proteins and their
application.

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Keywords:-
*Proteins
*Protein Identification
*Protein Structural Analysis
*Protein purification
*ELISA
*Protein Quantitation
*Western blotting

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PROTEINS EXPLAINED:

Proteins, also known as polypeptides, are organic compounds made up of amino


acids. They are arranged in a linear chain and folded into a globular form. Proteins
are essential parts of organisms and they participate in virtually every process
within cells. Many proteins are enzymes that catalyse biochemical reactions and
are vital to metabolism. The size of a protein is an important physical characteristic
and scientists often use particle size analysers in their studies to discuss protein
size or molecular weight.

THE STRUCTURE OF PROTEINS:

Most proteins fold into unique three-dimensional structures. The shape that a
protein folds into naturally is known as its native conformation. While most
proteins can fold unassisted through the chemical properties of their amino acids,
others require the aid of molecular chaperones. There are four distinct aspects of a
protein’s structure:

 Primary structure: the amino acid sequence


 Secondary structure: regularly repeating local structures stabilised by hydrogen
bonds
 Tertiary structure: the overall shape of a single protein molecule; the spatial
relationship of the secondary structures to one another
 Quatemary structure: the structure formed by several protein molecules which
function as a single protein complex

PROTEIN ANALYSIS TECHNIQUES:

Proteins differ from each other according to the type, number and sequence of
amino acids that make up the polypeptide backbone. Hence, they have different

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molecular structures, nutritional attributes and physiochemical properties. There


are three major protein analysis techniques, protein separation, western blotting
and protein identification.

1. PROTEIN SEPARATION:

 SDS-PAGE: This method separates proteins mainly on the basis of molecular


weight as opposed to charge or folding. It is a technique that is widely used in
biochemistry, forensics, genetics and molecular biology.
 Isoelectric Focusing: In this method, different molecules are separated by their
electric charge differences. This technique is a type of zone electrophoresis that is
usually performed in a gel and takes advantage of the fact that a molecule’s charge
changes with the pH of its surroundings.
 Chromatic Methods: There are two chromatic methods frequently used for
protein separation – high-performance liquid chromatography and thin-layer
chromatography. Both these methods are particularly useful adjuncts to gel-based
approaches. Although chromatography is a common technique in biochemistry
laboratories used for purification, identification and quantification of protein
mixtures, laser diffraction is traditionally used for pre-column size and
polydispersity management.
 Two-dimensional Gel Electrophoresis: This is a powerful gel-based method
commonly used to analyse complex samples in the interest of characterising the
full range of proteins in the sample, not just a few specific proteins.

2. WESTERN BLOTTING

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The most common version of this method is immunoblotting. This technique is


used to detect specific proteins in a given sample of tissue homogenate or extract.
The sample of proteins is first electrophoresed by SDS-PAGE to separate the
proteins based on molecular weight. The proteins are then transferred to a
membrane where they are probed using antibodies specific to the target protein.
EXPLANATION:

Western Blot (WB) is a common method to detect and analyze proteins. It is built
on a technique that involves transferring, also known as blotting, proteins separated
by electrophoresis from the gel to a membrane where they can be visualized
specifically. The procedure was first described by H. Towbin et al in 1979
(Towbin, Staehelin, & Gordon, 1979) and two years later given its name by W.
Neal Burnette (Burnette, 1981). Towbin et al described electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets where the original gel
pattern was accurately obtained. The setup consists of a standard set of seven steps,
Figure .

Figure. The standard steps in Western Blotting.

Samples are prepared and loaded on to a gel and during the electrophoresis the
negatively charged proteins move toward the positively charged anode. In order to
further analyze the proteins, they are transferred onto a membrane in a procedure
called blotting. After the transfer, the membrane is blocked in order to prevent
unwanted membrane-protein interaction in the following steps. To visualize the
protein of interest the membrane is commonly first probed using a primary protein-
specific antibody followed by a labeled secondary antibody used for detection. An
image is taken of the membrane and the result is analyzed.

HOW PROCESS OCCUR?


By adding a separate marker solution to one of the wells in the gel, it is possible to
estimate the size of the protein in addition to the antibody interactions that are used

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to verify the specific protein. The separation on the gel is not only due to size but
also to some extent depending on the molecular charge, hydrophobic regions, and
degree of denaturation. The setup of the experiment can be varied in many ways to
best suit the specific inquiry. When analyzing the results, variations between lanes
regarding loading and transfer rates between blots, must be taken into
consideration. In addition, the non-linear relation of the generated signal across the
concentration range of the samples is also an aspect of consideration when
interpreting the results. The outcome of a WB experiment depends on three
important factors; the ability of the antibody to bind a specific protein, the strength
of the interaction, and the concentration of the protein of interest itself. Moreover,
the specificity of the binding to the target and a low cross reactivity are important
features as well. The result form the WB is not always easy to interpret as the size
of the protein may vary from the theoretical weight due to posttranslational
modifications, such as glycosylation, or interactions with other proteins. However,
WB is a very common method and almost all available commercial antibodies
have been validated .

Figure 5. Typical Western Blot result using HRP and a CCD camera.

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3. PROTEIN IDENTIFICATION

There are two methods that are commonly used to identify proteins.

Edman Degradation: Developed by Pehr Edman, this is a method of sequencing


amino acids in a peptide. Here, the amino-terminal residue is labeled and cleaved
from the peptide without disrupting the peptide bonds between other amino acid
residues.

EXPLANATION:
The Edman degradation is a very important reaction for protein sequencing,
because it allows the ordered amino acid composition of a protein to be discovered.
Automated Edman sequencers are now in widespread use, and are able to sequence
peptides up to approximately 50 amino acids long. A reaction scheme for
sequencing a protein by the Edman degradation follows; some of the steps are
elaborated on subsequently.

1. Break any disulfide bridges in the protein with a reducing agent like 2-
mercaptoethanol. A protecting group such as iodoacetic acid may be
necessary to prevent the bonds from re-forming.
2. Separate and purify the individual chains of the protein complex, if there are
more than one.
3. Determine the amino acid composition of each chain.
4. Determine the terminal amino acids of each chain.
5. Break each chain into fragments under 50 amino acids long.
6. Separate and purify the fragments.
7. Determine the sequence of each fragment.
8. Repeat with a different pattern of cleavage.
9. Construct the sequence of the overall protein.

 Mass Spectrometry: An analytical technique that measures the mass-to-charge


ratio of charged particles, mass spectrometry is used for determining masses of
particles and the elemental composition of a sample of molecule as well as for
elucidating the chemical structure of molecules such as peptides.
PROTEIN SEQUENCING:

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 Protein sequencing is the practical process of determining the amino acid


sequence of all or part of a protein or peptide. This may serve to identify the
protein or characterize its post-translational modifications. Typically, partial
sequencing of a protein provides sufficient information (one or more
sequence tags) to identify it with reference to databases of protein sequences
derived from the conceptual translation of genes.
 The two major direct methods of protein sequencing are mass
spectrometry and Edman degradation using a protein
sequenator (sequencer). Mass spectrometry methods are now the most
widely used for protein sequencing and identification but Edman
degradation remains a valuable tool for characterizing a protein's N-
terminus.
It is often desirable to know the unordered amino acid composition of a protein
prior to attempting to find the ordered sequence, as this knowledge can be used to
facilitate the discovery of errors in the sequencing process or to distinguish
between ambiguous results. Knowledge of the frequency of certain amino acids
may also be used to choose which protease to use for digestion of the protein. The
misincorporation of low levels of non-standard amino acids (e.g. norleucine) into
proteins may also be determined. A generalized method often referred to as amino
acid analysis for determining amino acid frequency is as follows:

1. Hydrolyse a known quantity of protein into its constituent amino acids.


2. Separate and quantify the amino acids in some way.
Hydrolysis:
Hydrolysis is done by heating a sample of the protein in 6 M hydrochloric acid to
100–110 °C for 24 hours or longer. Proteins with many bulky hydrophobic groups
may require longer heating periods. However, these conditions are so vigorous that
some amino acids (serine, threonine, tyrosine, tryptophan, glutamine, and cysteine)
are degraded.
Separation and quantitation:
The amino acids can be separated by ion-exchange chromatography then
derivatized to facilitate their detection. More commonly, the amino acids are
derivatized then resolved by reversed phase HPLC.
An example of the ion-exchange chromatography is given by the NTRC using
sulfonated polystyrene as a matrix, adding the amino acids in acid solution and
passing a buffer of steadily increasing Ph through the column. Amino acids are
eluted when the pH reaches their respective isoelectric points.

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Gel electrophoresis is a method for separation and analysis of macromolecules


(DNA, RNA and proteins) and their fragments, based on their size and charge. It is
used in clinical chemistry to separate proteins by charge or size (IEF agarose,
essentially size independent) and in biochemistry and molecular biology to
separate a mixed population of DNA and RNA fragments by length, to estimate the
size of DNA and RNA fragments or to separate proteins by charge.

Nucleic acid molecules are separated by applying an electric field to move the
negatively charged molecules through a matrix of agarose or other substances.
Shorter molecules move faster and migrate farther than longer ones because shorter
molecules migrate more easily through the pores of the gel. This phenomenon is
called sieving. Proteins are separated by charge in agarose because the pores of the
gel are too large to sieve proteins. Gel electrophoresis can also be used for
separation of nanoparticles.
EXPLANATION:
Gel electrophoresis uses a gel as an anticonvective medium or sieving medium
during electrophoresis, the movement of a charged particle in an electrical field.
Gels suppress the thermal convection caused by application of the electric field,
and can also act as a sieving medium, retarding the passage of molecules; gels can
also simply serve to maintain the finished separation, so that a post electrophoresis
stain can be applied. DNA Gel electrophoresis is usually performed for analytical
purposes, often after amplification of DNA via polymerase chain reaction (PCR),
but may be used as a preparative technique prior to use of other methods such
as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or Southern
blotting for further characterization.

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Figure 3. The proteins in the gel are blotted to a membrane and the sample is
visualized through blocking, adding antibodies, and washing according to a certain
schedule.

HOW IT DIFFERNTIATED FROM OTHER TECHNIQUES?


After sample preparation the extract is ready to be loaded to separate the proteins
according to size by gel electrophoresis. An electric field is applied over the gel
that causes the charged molecules to move. In WB polyacrylamide gels are used
for protein separation and the method is therefore called polyacrylamide gel
electrophoresis (PAGE) when using native condition. For denaturing conditions,
sodium dodecyl sulfate (SDS) is added to the system and the method is therefore
called SDS-PAGE. The SDS binds to the protein and form a negatively charged
micelle around the protein regardless of inherent charge. The denaturing condition
dissolves the tridimensional structure of the proteins and the charge of the protein
becomes relative to its size resulting in separation of the proteins only by size.
When using native conditions the mobility is depending on both charge and
hydrodynamic size allowing detection of changes in charge due to chemical
degradation, conformational changes due to folding or unfolding, aggregation, or
other binding events.

TYPES OF GEL:

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The gel typically consists of two sections with different densities:


(i) a stacking gel
(ii) a separating gel.
The differences between the two sections are in pH and gel concentration. With
somewhat acidic pH and a lower concentration of acrylamide the stacking gel
separates the proteins poorly but allows them to form highly defined sharp bands
before they enter the separating gel. With more basic conditions and higher gel
concentration, the separating gel makes the proteins differentiate by size as smaller
proteins travel faster in the gel than bigger ones. Precast gels are convenient;
however, it is possible to cast them by hand. The gel is immersed in buffer and the
protein samples and markers are loaded to the wells in the gel. A voltage is applied
on the gel and the proteins will start to travel down the gel due to their negative
electrical charge. Selecting the proper voltage is important since too high voltage
will overheat the gel and maybe deform the bands.

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Figure 2. A typical gel immersed in buffer.

ELISA:

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique


designed for detecting and quantifying substances such as peptides, proteins,
antibodies and hormones. Other names, such as enzyme immunoassay (EIA), are
also used to describe the same technology.

PROCESS OCCURING:
In an ELISA, an antigen must be immobilized on a solid surface and then
complexed with an antibody that is linked to an enzyme. Detection is accomplished
by assessing the conjugated enzyme activity via incubation with a substrate to
produce a measureable product. The most crucial element of the detection strategy
is a highly specific antibody-antigen interaction.
ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which
passively bind antibodies and proteins. It is this binding and immobilization of
reagents that makes ELISAs so easy to design and perform. Having the reactants of
the ELISA immobilized to the microplate surface makes it easy to separate bound
from non-bound material during the assay. This ability to wash away
nonspecifically bound materials makes the ELISA a powerful tool for measuring
specific analytes within a crude preparation.

A detection enzyme or other tag can be linked directly to the primary antibody or
introduced through a secondary antibody that recognizes the primary antibody. It
can also be linked to a protein such as streptavidin if the primary antibody is biotin
labeled. The most commonly used enzyme labels are horseradish peroxidase
(HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but
they have not gained widespread acceptance because of limited substrate options.
These include β-galactosidase, acetylcholinesterase and catalase. A large selection
of substrates is available for performing ELISA with an HRP or AP conjugate. The
choice of substrate depends upon the required assay sensitivity and the
instrumentation available for signal-detection (spectrophotometer, fluorometer or
luminometer).

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MODIFICATIONS:
ELISAs can be performed with a number of modifications to the basic procedure.
The key step, immobilization of the antigen of interest, can be accomplished by
direct adsorption to the assay plate or indirectly via a capture antibody that has
been attached to the plate. The antigen is then detected either directly (labeled
primary antibody) or indirectly (labeled secondary antibody). The most powerful
ELISA assay format is the sandwich assay. This type of capture assay is called a
“sandwich” assay because the analyte to be measured is bound between two
primary antibodies – the capture antibody and the detection antibody. The
sandwich format is used because it is sensitive and robust.

Diagram of common ELISA formats (direct vs. sandwich assays). In the assay,
the antigen of interest is immobilized by direct adsorption to the assay plate or by
first attaching a capture antibody to the plate surface. Detection of the antigen can
then be performed using an enzyme-conjugated primary antibody (direct detection)
or a matched set of unlabeled primary and conjugated secondary antibodies
(indirect detection).

Comparison of direct and indirect ELISA detection methods

Direct ELISA detection

Advantages  Quick because only one antibody and fewer steps are used.
 Cross-reactivity of secondary antibody is eliminated.

Disadvantages  Immunoreactivity of the primary antibody might be adversely affected


by labeling with enzymes or tags.

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 Labeling primary antibodies for each specific ELISA system is time-


consuming and expensive.
 No flexibility in choice of primary antibody label from one experiment
to another.
 Minimal signal amplification.

Indirect ELISA detection

Advantages  A wide variety of labeled secondary antibodies are available


commercially.
 Versatile because many primary antibodies can be made in one species
and the same labeled secondary antibody can be used for detection.
 Maximum immunoreactivity of the primary antibody is retained
because it is not labeled.
 Sensitivity is increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary antibody, allowing
for signal amplification.
 Different visualization markers can be used with the same primary
antibody.

Disadvantages  Cross-reactivity might occur with the secondary antibody, resulting in


nonspecific signal.
 An extra incubation step is required in the procedure.

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REFERNCES:
1. Voet, D. and J. G.Voet. 2011. Biochemistry, 4th Edition. John Wiley and Sons. Inc.,
New York.

2. David L. Nelson Michael M. Cox. 2008. Lehninger Analytical Chemistry, 5h


Edition. W.H. Freeman and Company, USA.

3. Campbell, M.K. and S.O. Farrell. 2006. 5th Edition.Biotechnolgy Techniques. Baba
BarkhaNath Printers, Delhi.

4. Nigam, A. and A. Ayyagari. 2008. Lab Manual in Biochemistry and


Biotechnology. McGraw-Hill and Companies, New Delhi.

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