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Biochemical Analysis Techniques

Biochemical analysis techniques

Biochemical analysis techniques refer to a set of methods,

assays, and procedures that enable scientists to analyze the
substances found in living organisms and the chemical
reactions underlying life processes. The most sophisticated of
these techniques are reserved for specialty research and
diagnostic laboratories, although simplified sets of these
techniques are used in such common events as testing for illegal drug abuse in competitive
athletic events and monitoring of blood sugar (/medicine/anatomy-and-physiology/anatomy-
and-physiology/blood-sugar) by diabetic patients.

To perform a comprehensive biochemical analysis of a biomolecule in a biological process or

system, the biochemist typically needs to design a strategy to detect that biomolecule, isolate it
in pure form from among thousands of molecules that can be found in an extracts from a
biological sample, characterize it, and analyze its function. An assay, the biochemical test that
characterizes a molecule, whether quantitative or semi-quantitative, is important to determine
the presence and quantity of a biomolecule at each step of the study. Detection assays may
range from the simple type of assays provided by spectrophotometric measurements and gel
staining to determine the concentration and purity of proteins and nucleic acids, to long and
tedious bioassays that may take days to perform.

The description and characterization of the molecular components of the cell succeeded in
successive stages, each one related to the introduction of new technical tools adapted to the
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particular properties of the studied molecules. The first studied biomolecules were the small
building blocks of larger and more complex macromolecules, the amino acids of proteins, the

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bases of nucleic acids and sugar monomers of complex carbohydrates. The molecular
characterization of these elementary components was carried out thanks to techniques used in
organic chemistry (/science-and-technology/chemistry/organic-chemistry/organic-chemistry)
and developed as early as the nineteenth century. Analysis and characterization of complex
macromolecules proved more difficult, and the fundamental techniques in protein and nucleic
acid and protein purification and sequencing were only established in the last four decades.

Most biomolecules occur in minute amounts in the cell, and their detection and analysis
require the biochemist to first assume the major task of purifying them from any
contamination . Purification procedures published in the specialist literature are almost as
diverse as the diversity of biomolecules and are usually written in sufficient details that they
can be reproduced in different laboratory with similar results. These procedures and protocols,
which are reminiscent of recipes in cookbooks have had major influence on the progress of
biomedical sciences and were very highly rated in scientific literature.

The methods available for purification of biomolecules range from simple precipitation,
centrifugation, and gel electrophoresis to sophisticated chromatographic and affinity
techniques that are constantly undergoing development and improvement. These diverse but
interrelated methods are based on such properties as size and shape, net charge and
bioproperties of the biomolecules studied.

Centrifugation procedures impose, through rapid spinning, high centrifugal forces on

biomolecules in solution, and cause their separations based on differences in weight.
Electrophoresis techniques take advantage of both the size and charge of biomolecules and
refer to the process where biomolecules are separated because they adopt different rates of
migration toward positively (anode) or negatively (cathode) charged poles of an electric field.
Gel electrophoresis methods are important steps in many separation and analysis techniques
in the studies of DNA , proteins and lipids. Both western blotting techniques for the assay of
proteins and southern and northern analysis of DNA rely on gel electrophoresis. The
completion of DNA sequencing at the different human genome centers is also dependent on
gel electrophoresis. A powerful modification of gel electrophoresis called twodimensional gel
electrophoresis is predicted to play a very important role in the accomplishment of the
proteome projects that have started in many laboratories.
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Chromatography techniques are sensitive and effective in separating and concentrating minute
components of a mixture and are widely used for quantitative and qualitative analysis in
medicine, industrial processes, and other fields. The method consists of allowing a liquid or
gaseous solution of the test mixture to flow through a tube or column packed with a finely
divided solid material that may be coated with an active chemical group or an adsorbent liquid.
The different components of the mixture separate because they travel through the tube at
different rates, depending on the interactions with the porous stationary material. Various
chromatographic separation strategies could be designed by modifying the chemical
components and shape of the solid adsorbent material. Some chromatographic columns used
in gel chromatography are packed with porous stationary material, such that the small
molecules flowing through the column diffuse into the matrix and will be delayed, whereas
larger molecules flow through the column more quickly. Along with ultracentrifugation and gel
electrophoresis, this is one of the methods used to determine the molecular weight (/science-
and-technology/chemistry/chemistry-general/molecular-weight) of biomolecules. If the
stationary material is charged, the chromatography column will allow separation of
biomolecules according to their charge, a process known as ion exchange chromatography.
This process provides the highest resolution in the purification of native biomolecules and is
valuable when both the purity and the activity of a molecule are of importance, as is the case
in the preparation of all enzymes used in molecular biology (/science-and-
technology/biology-and-genetics/cell-biology/molecular-biology) . The biological activity
of biomolecules has itself been exploited to design a powerful separation method known as
affinity chromatography. Most biomolecules of interest bind specifically and tightly to natural
biological partners called ligands: enzymes bind substrates and cofactors, hormones bind
receptors, and specific immunoglobulins called antibodies can be made by the immune
system that would in principle interact with any possible chemical component large enough to
have a specific conformation. The solid material in an affinity chromatography column is
coated with the ligand and only the biomolecule that specifically interact with this ligand will be
retained while the rest of a mixture is washed away by excess solvent running through the
column.

Once a pure biomolecule is obtained, it may be employed for a specific purpose such as an
enzymatic reaction, used as a therapeutic agent, or in an industrial process. However, it is
normal in a research laboratory that the biomolecule isolated is novel, isolated for the first time ×
and, therefore, warrants full characterization in terms of structure and function. This is the most

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difficult part in a biochemical analysis of a novel biomolecule or a biochemical process, usually

takes years to accomplish, and involves the collaboration of many research laboratories from
different parts of the world.

Recent progress in biochemical analysis techniques has been dependant upon contributions
from both chemistry and biology, especially molecular genetics and molecular biology
(/science-and-technology/biology-and-genetics/cell-biology/molecular-biology), as well as
engineering and information technology. Tagging of proteins and nucleic acids with chemicals,
especially fluorescent dyes , has been crucial in helping to accomplish the sequencing of the
human genome and other organisms, as well as the analysis of proteins by chromatography
and mass spectrometry. Biochemical research is undergoing a change in paradigm from
analysis of the role of one or a few molecules at a time, to an approach aiming at the
characterization and functional studies of many or even all biomolecules constituting a cell and
eventually organs. One of the major challenges of the post-genome era is to assign functions
to all of the gene products discovered through the genome and cDNA sequencing efforts. The
need for functional analysis of proteins has become especially eminent, and this has led to the
renovated interest and major technical improvements in some protein separation and analysis
techniques. Two-dimensional gel electrophoresis, high performance liquid and capillary
chromatography as well as mass spectrometry are proving very effective in separation and
analysis of abundant change in highly expressed proteins. The newly developed hardware and
software, and the use of automated systems that allow analysis of a huge number of samples
simultaneously, is making it possible to analyze a large number of proteins in a shorter time
and with higher accuracy. These approaches are making it possible to study global protein
expression in cells and tissues, and will allow comparison of protein products from cells under
varying conditions like differentiation and activation by various stimuli such as stress,
hormones, or drugs. A more specific assay to analyze protein function in vivo is to use
expression systems designed to detect protein-protein and DNA-protein interactions such as
the yeast and bacterial hybrid systems. Ligand-receptor interactions are also being studied by
World of Microbiology and Immunology
novel techniques using biosensors that are much faster than the conventional

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