In the past the analysis of proteins was a tedious, time consuming procedure. Recently in biochemical analysis, the importance of effective and efficient studies of proteins, such as protein expression, detection and identification has been a major interest in the area of proteomic and diagnostic research.( As different kinds of diseases are linked to certain proteins and certain irregularities in protein structures and their expression in biochemical reactions in the body)
"This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNAS."(1.1) However the use of traditional protein analysis is quite time consuming. I.e. -- the use of methods such as western blotting, 2D electrophoresis and the use of a reporter gene. This is why it is important to develop methods of analysing proteins in a more high through-put fashion which allows proteins to be analysed quickly, in large amounts and in a way which is direct.
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One method which has been developed is the use of protein microarrays. A protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. (1)
The main advantage and difference in this method compared to previously used methods lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a surface used for support for example such as a glass slide, bead, or microtitre plate. Through using these support surfaces an array of captured proteins can be bound.
"The development of protein microarrays makes possible interaction-based protein assays in miniaturised, multiplexed formats. A major requirement determining their uptake and use is the availability and stability of purified, functional proteins for immobilisation" (2)
Probe molecules, which are usually labelled with a fluorescent dye, are added also to the array. If a reaction between the probe molecule and protein occurs then a fluorescent light is released that is read by a scanner. Protein microarrays are fast, mechanical, and highly sensitive and economical in the sense that they can run on small amounts of reagents and materials.
The high-throughput technology behind the protein microarray was quite easy to develop as it was based on the general idea of the already developed DNA microarray technology. These were an extreme advance in the biochemical business and a lot of the technology involved in DNA libraries such as PCR, and hybridization have come together in the production of DNA microarrays. These allowed the genome to be processed rapidly whilst being screened simultaneously.
Proteins can also be immobilised on a solid surface to define the presence or absence of proteins in a sample. Many of these protein microarrays have been developed as a high throughput method of protein analysis and are used in many fields such as protein studies, diagnostics and in drug screening processes. This review will cover the basic applications of protein microarrays and their importance in the biochemical business. It will also cover the technique involved in the production of these usefully microarrays. (3) A hands on example of this is scientists can prepare an array of antibodies which are linked to certain proteins by immobilizing them as single areas in the on a solid surface. A sample of proteins is then mixed and if the protein which combines with any of the antibodies present in the sample. It can then be detected by a solid state form of the ELISA assay. (Enzyme-linked immunosorbent assay)
The small spots have many essential advantages when compared with the use of larger spots in protein microarrays. The reason for this is that they require less time to reach equilibrium in processes such as binding and washing, increased signals due to reduced depletion of probe materials, larger spectrum of detection, techniques and better compatibility with electronic microchips.(4) The small spots also increase rate of heat exchange during the production and requirements for field homogeneity. Many other types and applications of protein microarrays are being developed which will prove to be promising as protein microarray technology is in its infancy. (5)
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Proteins can be also attached onto the intrinsically charged surface of a substrate or a surface modified with charged molecules such as poly- lysine, using electro static interaction. The non- covalent end of a protein using hydrophobic bonding may result in the denaturation of the protein by causing the protein to un-fold. It also may cause the protein to become weakened for trying to retain stability in an assay procedure due to the fact that the non-covalent ends attached to it can be reversed.
Therefore, proteins are required for immobilization along with covalent bonding to increase the stabilisation of immobilized protein and the manipulation of availability of the site where the protein binds. The use of a modified surface of the substrates to reactive molecules, such as aldehydes, and esters make covalent linkage with amine or carboxyl end of a targeted protein may cause the effectiveness of protein immobilization to be improved.
There are three types of protein microarrays that are currently used in proteomics.
Analytical microarrays, these are also known as capture arrays because of their method.
In analytical microarrays, there is a library of antibodies which are burned on the support surface which is usually and commonly glass. These antibodies act as molecules which capture the protein since each combines specifically to a certain protein. The array is introduced with a protein solution which is quite complex. An example of such a solution is cell lysate.
The analysis of the binding reactions which occur can be done using various detection systems which can supply researchers with information involving the expression levels of certain proteins in the sample along with figures for their affinity in terms of binding.
Functional protein microarrays, these are also known as target protein arrays.
These protein microarrays are produced by immobilising large numbers of purified proteins and are used to identify protein, protein involved in DNA, protein involved in RNA, protein involved with phospholipids, and protein-small molecule encounters, to assay enzymatic activity and to detect antibodies and demonstrate their specificity. These microarrays are different from analytical arrays (As mentioned above) because these arrays are made up of full length proteins. These protein chips are used to study the biochemical activities of the entire proteome in a single experiment.
Reverse phase protein microarray
These protein microarrays are involved with sample of great complexity. An example of such would be tissue cell lysate. The cells in the tissue are isolated from different kind of tissues which are of interest and are cut. The lysate is arrayed onto the microarray and probed with antibodies against the target protein of interest. These antibodies are usually detected through the use of chemiluminescent, fluorescent. (See figure 1.)Molecules known as reference peptides are printed on the support surface to allow the analysis of the protein and to calculate quantification of the sample. RPA's are used to determine the possible presence of proteins which have been altered in some way which in turn may be used to diagnosis diseases or give researchers an understanding of what cause diseases.
Theses protein chips can be used in many areas of biochemistry but there are 5 main areas in which they can be applied. The 5 main areas are diagnostics, proteomics, protein function analysis, antibody characterization and treatment development.
In diagnostics the protein micro arrays can be used to help detection of antigens and antibodies in the blood. It can also be used as method of testing how the body is responding to therapy. Several diagnostic protein microarray products have been cleared by the US Food and Drug Administration (FDA). The diagnosis of autoimmune disease is the focus of most of these proteins microarrays including the AtheNA Multi-Lyte Test system and the Bio-Plex 2200 system. (6)
In proteomics the protein microarrays can be used in protein profiling. The protein microchip can also be used in protein functional analysis. They can be used in identifying interactions between proteins and their ligands. It can be used in antibody characterisation. This can be used in characterising the reactivity of antibodies and also can be used in the mapping of epitopes.
It can be used in treatment development and in therapies for autoimmune diseases. Using the microarray technology could allow the identification of small molecules which could potentially be used to manufacture new drugs which could be used to tackle the problem of cancer.
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The problem with protein microarrays is they are quite difficult to handle when compared with DNA microchips. There is a few challenges involved which if tackle could lead to a breakthrough in the biochemical department and in diagnosis of diseases and possible help in the curing of diseases.
These problems include finding a suitable surface and a method which allows the protein to maintain their structure and their biologically activity and their encounters with other molecules. There is also need the production of an array which has a long shelf life as proteins generally denatures on the chips after a small amount of time. It is also required to have the non-specific binding by the capturing agents.
Although the technology is still in its infancy it is constantly becoming more and more advance and in a few years the technology may be able to provide ground breaking diagnosis of diseases which may even be linked with the abolishing of terminal diseases such as cancer or even provide the information needed to understand how and why proteins are mutated to cause these diseases. All that is needed is a push to further our research into the field of microarrays.