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Protein Microarrays
Chien-Sheng Chen Heng Zhu
Department of Pharmacology and Molecular Sciences/High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
BioTechniques, Vol. 40, No. 4, April 2006, pp. 423–429
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Protein microarrays, an emerging class of proteomic technologies, are fast becoming critical tools in biochemistry and molecular biology. Two classes of protein microarrays are currently available: analytical and functional protein microarrays. Analytical protein microarrays, mostly antibody microarrays, have become one of the most powerful multiplexed detection technologies. Functional protein microarrays are being increasingly applied to many areas of biological discovery, including studies of protein interaction, biochemical activity, and immune responses. Great progress has been achieved in both classes of protein microarrays in terms of sensitivity, specificity, and expanded application.

Protein microarrays, also known as protein chips, are miniaturized and parallel assay systems that contain small amounts of purified proteins in a high-density format (1). They allow simultaneous determination of a great variety of analytes from small amounts of samples within a single experiment. Protein microarrays are typically prepared by immobilizing proteins onto a microscope slide using a standard contact spotter (1,2) or noncontact microarrayer (3,4,5). A variety of slide surfaces can be used. Popular types include aldehyde-and epoxy-derivatized glass surfaces for random attachment through amines (2,6), nitrocellulose (7,8), or gel-coated slides (9,10) and nickel-coated slides for affinity attachment of His6-tagged proteins. The last type was reported to provide 10-fold better signals than those obtained with other random attachment methods (1). After proteins are immobilized on the slides, they can be probed for a variety of functions/ activities. Finally, the resulting signals are usually measured by detecting fluorescent or radio-isotope labels. The typical image of protein microarrays is shown as (Figure 1).

Figure 1.

A typical protein microarray image. A yeast protein microarray is probed with anti-GST antibodies followed by detection with Cy5-conjugated secondary antibodies. An enlarged image of one of the 48 blocks is depicted below the protein chip.

Analytical protein arrays can be used to monitor protein expression levels or for bio-marker identification, clinical diagnosis, or environmental/food safety analysis. Functional protein microarrays have many uses: (i) to probe for various types of protein activities, including protein-protein, protein-lipid, protein-DNA, protein-drug, and protein-pep-tide interactions; (ii) to identify enzyme substrates; and (iii) to profile immune responses, among many others. Applications of both the analytical and functional protein microarrays are depicted in (Figure 2). In the following sections, we will provide examples of various applications of both types of microarray, with an emphasis on functional protein microarrays. Given the large volume of papers related to protein microarray technology, we regret that we are unable to cite all the published work in the field.

Figure 2.

Applications of protein microarrays. Antibody arrays can be used for clinical diagnosis or environmental/food safety analysis. Functional protein arrays are mainly used to study various types of protein activities, including protein-protein, protein-lipid, protein-DNA, protein-drug, and protein-peptide interactions, to identify enzyme substrates and to profile immune responses.

Analytical Microarrays

Perhaps the most representative class of analytical micro-arrays is the antibody microarray, in which antibodies are arrayed on glass surfaces at high density. The biggest challenge associated with antibody microarrays is that of producing antibodies that are able to identify the proteins of interest with high specificity and affinity in a high-throughput fashion. Because the traditional method for generating monoclonal antibodies is time-consuming and laborious, researchers have recently sought alternative approaches. For example, phage antibody-display, ribosome display, systematic evolution of ligands by exponential enrichment (SELEX), messenger RNA (mRNA) display, and affibody display have been developed to expedite the production of antibodies with high specificity (11,12,13,14). All of these methods involve the construction of large repertoires of viable regions with potential binding activity, which can be selected by multiple rounds of affinity purification. The binding affinity of the resulting candidate clones can be further improved using maturation strategies. However, the ideal selection system is yet to be fully developed: one that is not only fast, robust, sensitive, and of low cost, but also automated and minimized (13,14).

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