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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
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.
Keywords:-
*Proteins
*Protein Identification
*Protein Structural Analysis
*Protein purification
*ELISA
*Protein Quantitation
*Western blotting
PROTEINS EXPLAINED:
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:
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
1. PROTEIN SEPARATION:
2. WESTERN BLOTTING
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 .
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.
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.
3. PROTEIN IDENTIFICATION
There are two methods that are commonly used to identify proteins.
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.
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.
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.
TYPES OF GEL:
ELISA:
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).
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).
Advantages Quick because only one antibody and fewer steps are used.
Cross-reactivity of secondary antibody is eliminated.
REFERNCES:
1. Voet, D. and J. G.Voet. 2011. Biochemistry, 4th Edition. John Wiley and Sons. Inc.,
New York.
3. Campbell, M.K. and S.O. Farrell. 2006. 5th Edition.Biotechnolgy Techniques. Baba
BarkhaNath Printers, Delhi.