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 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 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 techniques used in 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.
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 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 . 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 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, 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 novel techniques using biosensors that are much faster than the conventional immunochemical and colorimetric analyzes.
The combination of large scale and automated analysis techniques, bioinformatic tools, and the power of genetic manipulations will enable scientists to eventually analyze processes of cell function to all depths.
See also Bioinformatics and computational biology; Biotechnology; Fluorescence in situ hybridization; Immunological analysis techniques; Luminescent bacteria
"Biochemical Analysis Techniques." World of Microbiology and Immunology. . Encyclopedia.com. (August 4, 2019). https://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/biochemical-analysis-techniques
"Biochemical Analysis Techniques." World of Microbiology and Immunology. . Retrieved August 04, 2019 from Encyclopedia.com: https://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/biochemical-analysis-techniques