An aqueous environment is essential at all stages of array manufacture and operation to prevent protein denaturation. Therefore, sample buffers contain a high percent of glycerol to lower the freezing point , and the humidity of the manufacturing environment is carefully regulated. Microwells have the dual advantage of providing an aqueous environment while preventing cross-contamination between samples.
In the most common type of protein array, robots place large numbers of proteins or their ligands onto a coated solid support in a pre-defined pattern. This is known as robotic contact printing or robotic spotting. Another fabrication method is ink-jetting , a drop-on-demand, non-contact method of dispersing the protein polymers onto the solid surface in the desired pattern. The printhead moves across the array, and at each spot uses electric stimulation to deliver the protein molecules onto the surface via tiny jets. This is also a non-contact process. Light is used in association with photomasks , opaque plates with holes or transparencies that allow light to shine through in a defined pattern.
A series of chemical treatments then enables deposition of the protein in the desired pattern upon the material underneath the photomask. The capture molecules arrayed on the solid surface may be antibodies , antigens , aptamers nucleic acid-based ligands , affibodies small molecules engineered to mimic monoclonal antibodies , or full length proteins. Sources of such proteins include cell-based expression systems for recombinant proteins , purification from natural sources, production in vitro by cell-free translation systems , and synthetic methods for peptides.
Many of these methods can be automated for high throughput production but care must be taken to avoid conditions of synthesis or extraction that result in a denatured protein which, since it no longer recognizes its binding partner, renders the array useless. Proteins are highly sensitive to changes in their microenvironment. This presents a challenge in maintaining protein arrays in a stable condition over extended periods of time.
In situ methods — invented and published by Mingyue He and Michael Taussig in   — involve on-chip synthesis of proteins as and when required, directly from the DNA using cell-free protein expression systems. Since DNA is a highly stable molecule it does not deteriorate over time and is therefore suited to long-term storage. This approach is also advantageous in that it circumvents the laborious and often costly processes of separate protein purification and DNA cloning , since proteins are made and immobilised simultaneously in a single step on the chip surface.
There are three types of protein microarrays that are currently used to study the biochemical activities of proteins.
Microarrays in Drug Discovery and Development
Analytical microarrays are also known as capture arrays. In this technique, a library of antibodies, aptamers or affibodies is arrayed on the support surface. These are used as capture molecules since each binds specifically to a particular protein. The array is probed with a complex protein solution such as a cell lysate. Analysis of the resulting binding reactions using various detection systems can provide information about expression levels of particular proteins in the sample as well as measurements of binding affinities and specificities.
This type of microarray is especially useful in comparing protein expression in different solutions.
Protein microarray technology: Assisting personalized medicine in oncology (Review)
For instance the response of the cells to a particular factor can be identified by comparing the lysates of cells treated with specific substances or grown under certain conditions with the lysates of control cells. Another application is in the identification and profiling of diseased tissues. Reverse phase protein microarray RPPA involve complex samples, such as tissue lysates.
Cells are isolated from various tissues of interest and are lysed. The lysate is arrayed onto the microarray and probed with antibodies against the target protein of interest. These antibodies are typically detected with chemiluminescent , fluorescent or colorimetric assays. Reference peptides are printed on the slides to allow for protein quantification of the sample lysates.
RPAs allow for the determination of the presence of altered proteins or other agents that may be the result of disease. Specifically, post-translational modifications, which are typically altered as a result of disease can be detected using RPAs. Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis.
Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein—protein interaction analysis, vaccine development, enzyme—substrate profiling, and immuno-profiling.
While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: 1 inconsistent printed microspot morphologies and uniformities, 2 low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, 3 inconsistent quantification of captured signal due to spot uniformity issues, 4 non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly 5 poor protein e.
Conventional printing approaches, including contact e. This review critiques current protein-based microarray preparation techniques commonly used for analytical and function-based proteomics and their effects on array-based assay performance. The article was received on 19 Aug , accepted on 15 Jan and first published on 16 Jan If you are not the author of this article and you wish to reproduce material from it in a third party non-RSC publication you must formally request permission using Copyright Clearance Center.
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