Biochemical analysis techniques WORLD OF MICROBIOLOGY AND IMMUNOLOGY64•
typically needs to design a strategy to detect that biomole-
cule, isolate it in pure form from among thousands of mole-
cules 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 quan-
titative 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 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 bases of nucleic acids and sugar monomers
of complex carbohydrates. The molecular characterization of
these elementary components was carried out thanks to tech-
niques used in organic chemistry and developed as early as
the nineteenth century. Analysis and characterization of com-plex macromolecules proved more difficult, and the funda-
mental 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 spe-
cialist literature are almost as diverse as the diversity of bio-
molecules and are usually written in sufficient details that
they can be reproduced in different laboratory with similar
results. These procedures and protocols, which are reminis-
cent 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 elec-
trophoresis 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 bio-
properties of the biomolecules studied.
Centrifugation procedures impose, through rapid spin-
ning, 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 bio-
molecules 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 tech-
niques 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 two-
dimensional gel electrophoresis is predicted to play a very
important role in the accomplishment of the proteome projects
that have started in many laboratories.
Chromatography techniques are sensitive and effective
in separating and concentrating minute components of a mix-
ture and are widely used for quantitative and qualitative analy-
sis 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 compo-
nents of the mixture separate because they travel through the
tube at different rates, depending on the interactions with the
porous stationary material. Various chromatographic separa-
tion 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 andTechnician performing biochemical analysis.womi_B 5/6/03 1:09 PM Page 64