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Protein Microarrays: Lining Up To Be Counted
Buying Tips
Dec 11 '07
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Introduction |
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| Protein analysis has come a long way in the last decade. No longer must researchers toil over repeated careful washings of affinity columns in the hopes of isolating their protein of interest, or showing its interaction with another protein. The advent of protein microarrays now gives scientists another option—one that’s faster, easier, and gives more results in one go.
Generally, a protein microarray is a piece of glass, such as a standard microscope slide, onto which an array of proteins has been affixed in a grid-like pattern. Antibody microarrays are common, and are used to pull proteins out of cell lysate solutions, for example. But protein microarrays can also consist of probes for protein-protein interactions, and probes to identify substrates of protein kinases. Protein microarrays have the advantages of their nucleic acid cousins—they are easily adaptable to high-throughput experiments, and allow one to interrogate many different samples simultaneously. Here is a sampler of some of the protein microarray products on offer and under development today. |
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New hardware |
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| Primorigen Biosciences is trying to reduce your microarray costs by using ordinary glass slides instead of high-grade glass. As Chuck Oehler, chairman and president of Primorigen, says: “Primorigen Biosciences is currently developing ‘Spots on Dots™’, a novel array platform to support protein/antibody microarrays and complement existing glass slides.” Taking advantage of materials with different hydrophobic and hydrophilic properties, the Spots on Dots™ microarrays will be made without frames, which can be “costly, unreliable, and cumbersome,” according to Oehler. “Our frameless arrays will enable direct quantitative detection of native proteins using reduced sample volumes (1-2 uL) for, among other things, the rapid selection and biophysical characterization of antibodies, including matched pairs; the measurement of dissociation constants between an antibody and several proteins simultaneously; the measurement of protein binding parameters, including time course of binding and competition analysis; and performing assays in 96 or 384 grids using either a fluorescent or colorimetric read-out.”
Oehler says that, “a challenge in the area of antibody microarrays is the availability of high-quality matched pairs that recognize native protein. We see low-cost array technology such as Primorigen's Spots On Dots™ emerging as a tool to rapidly identify and characterize matched pair antibodies. This will allow microarray content development to keep pace with target identification in the related fields of research.”
If you are particularly interested in human proteins, consider Invitrogen’s new Version 4.0 ProtoArray® Human Protein Microarray, which consists of more than 8,000 full-length human proteins arrayed in duplicate on a 1” × 3” ultra-thin nitrocellulose-coated slide. “This new version has even more relevant content compared to previous versions, including over 1,000 membrane-bound proteins, 378 kinases, and 380 druggable proteins,” says Jennifer Cannon, business area manager for ProtoArray at Invitrogen. “The ProtoArray® Human Protein Microarray is being utilized by both academic and pharmaceutical researchers for discovery of cancer and autoimmune disease biomarker discovery, identification of putative targets of orphan compounds, assessing mechanism of action for new drug compounds, and mapping kinase and ubiquitin ligases to their substrates.”
Cannon explains why Invitrogen believes that their system delivers higher quality microarrays compared to their competition: “Proteins are derived from sequence-validated open reading frames selected from Invitrogen’s Ultimate™ ORF Clone Collection. These full-length proteins are expressed as N-terminal GST fusion proteins using a baculovirus-based expression system. All proteins are purified under non-denaturing conditions and printed at +4°C to preserve native structure and proper functionality.” She maintains that because the proteins on Invitrogen’s microarrays were purified under non-denaturing conditions, they are more likely to be pure, properly folded proteins that behave as expected.
GenTel BioSciences offers their new APiX™ Cancer Biomarker Array Kit, among other new protein microarray products. The kit is a single capture antibody array and protein labeling system for profiling the relative abundance of nearly 50 different cancer-related proteins in eight serum samples simultaneously. “The foremost differentiator of GenTel products and services in the marketplace is the level of validation and quality control that we perform on our microarrays,” says Jared Browning, marketing manager at GenTel Biosciences. “GenTel is the first and only company, for example, to demonstrate that single capture antibody arrays (like the APiX™ Cancer Biomarker Array) can be engineered to achieve detectable and highly specific capture |
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Binding to sticky molecules |
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| For the microarrays to function properly, there must be some sort of sticky molecules anchored to the slide or other substrate that also captures the protein targets (for example, from a cell lysate solution). Primorigen is developing families of matched-pair monoclonal antibodies that “will be placed on microarrays to support research in areas including stem cell biology, regenerative medicine, and pancreatic beta-cell therapy (type I diabetes),” says Oehler. “These antibodies are undergoing thorough and rigorous screening to ensure they recognize native proteins in cell lysates. Many antibodies on the market today are only validated with recombinant antigen and denatured protein and will often not bind native target protein in solution.”
In contrast to using antibodies for protein capture on microarrays, ProteinOne relies on non-antibody purified “active” proteins to capture other proteins through interactions on the microarray. According to Joy Limpuangthip, director of marketing and business development at ProteinOne, “by using these purified active proteins, ProteinOne's scientists have developed the Active Protein Array (APA) system, a high-throughput method for studying protein expression, function and interaction.” ProteinOne also uses the Active Protein Array method to detect specific interactions between two proteins, and between proteins and other small molecules or nucleotides.
The Active Protein Array Transcription Array Kit is designed for the multiplex detection and interaction assessment of 36 activated transcription factors. This kit uses full length, purified, biologically active proteins on a nitrocellulose membrane. A radiolabeled protein or DNA probe is then used as bait to search for interactions with the immobilized proteins. The proteins comprising ProteinOne’s microarrays are also available for order individually, allowing you to verify results obtained with the Active Protein Array system using another, independent method. |
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Challenges and future developments |
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| Despite their obvious utility and ability to increase productivity in the lab, there are some obstacles to using protein microarrays that must be overcome, such as perception, according to Browning. “One challenge involved in using protein microarrays has been the lingering market perception that these products aren’t yet proven or that they generally fail to perform,” he says. “This ‘stigma’ is generally believed to have arisen as a result of the first generation of protein microarrays developed years ago, which were often plagued by sub-par performance in terms of accuracy, precision, selectivity, and sensitivity.” To address this concern, GenTel is developing “next generation” protein microarrays that meet or exceed the accepted criteria for these parameters.
Browning suggests another obstacle to widespread adoption of protein microarrays: the cost and availability of appropriate instrumentation and software to the end-users who are, in his opinion, most naturally suited to use them—ELISA users. “We see the future bringing lower cost instrumentation and software that makes using protein microarrays look and feel more like processing ELISAs, with the added benefit of obtaining more data points per sample,” says Browning. “In addition, we see an increase in the outsourcing of testing to companies like GenTel BioSciences for processing samples using protein microarrays.”
Oehler agrees that the cost of current protein microarray systems needs to come down before the wider adoption of this technology will take off. “Many of the available microarrays utilize fluorescent read-out,” he says. “While there are some advantages to this system, it requires costly and specialized equipment. We see the introduction of systems allowing flexible read-out and data collection, the adoption of highly sensitive colorimetric detection, and the use of lower cost general equipment for microarray imaging (e.g. flatbed scanners, CCD imaging).”
Finally, as protein microarrays become more widespread, there is more demand for different types of protein contents on the microarrays. With movement of the technique into clinical environments, where it could potentially revolutionize the treatment of individuals with personalized medicine, there is increasing demand for protein contents with relevance to disease markers and “druggability.”
“We continue to respond to customer requests for increased membrane-bound and ‘druggable’ protein content, in addition to representing a more significant portion of the proteome,” says Cannon. “As our clinical researcher base continues to grow, we will work to increase the autoantigen and cancer antigen content on future versions of the ProtoArray.” Cannon adds that Invitrogen is interested in nanolipid technologies that would allow for proper scaffolding of membrane-bound proteins—species for which it is notoriously difficult to screen. Hopefully some of these new products, or other similar ones, will help to accelerate your research along the right track to success.
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Caitlin Smith
Contributing Writer
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