Microarrays function-based

The CBM based microarray technology described by some authors offer fundamental advantages over current non-DNA microarray technology, such as retention of protein functionality after immobilization, ease of fabrication, extended stability of the printed microarray, integrated test for quality control (QC) and the capacity to print test proteins without a purification step [55, 169, 170]. These features, together with the intrinsic spedfidly of CBMs for individual carbohydrates and the fadle modification with peptides and fluorescent molecules, allow for efficient production of protein and peptide microarrays. These can be used in a variety of potential applications technically impractical via conventional microarray technologies [55]. 

Reverse genetic approaches start with a sequence and attempt to identify a function for the gene. This can be done by using PCR-based screens on DNA from insertionally mutagenized populations, ectopic misexpression of the gene, and expression studies, e.g., with RT-PCR or microarray analyses. 

The current practice of using a T-cell-dependent antibody response or natural killer cell function is based on analyses suggesting that these parameters would suffice to identify the vast majority of immunotoxicants.12 However, it is clear that there are exceptions12 and that some immunotoxicants are not effectively identified by these functional tests or by histopathology.3 Thus, it remains possible, particularly if the cost of microarray analysis decreases, that microarrays will eventually prove to be the most practical method available to identify immunotoxicants. 

A dendrimeric system with Cy3 and Cy5 as the reporter dyes was applied to develop a microarray-based technology for high-throughput functional genomics research [73],... 

In a different approach, fluorescence-based DNA microarrays are utilized (88). In a model study, chiral amino acids were used. Mixtures of a racemic amino acid are first subjected to acylation at the amino function with formation of A-Boc protected derivatives. The samples are then covalently attached to amine-functionalized glass slides in a spatially arrayed manner (Fig. 10). In a second step, the uncoupled surface amino functions are acylated exhaustively. The third step involves complete deprotection to afford the free amino function of the amino acid. Finally, in a fourth step, two pseudo-Qn nX. om.Qx c fluorescent probes are attached to the free amino groups on the surface of the array. An appreciable degree of kinetic resolution in the process of amide coupling is a requirement for the success of the ee assay (Horeau s principle). In the present case, the ee values are accessible by measuring the ratio of the relevant fluorescent intensities. About 8000 ee determinations are possible per day, precision amounting to +10% of the actual value ((S(S). Although it was not explicitly demonstrated that this ee assay can be used to evaluate enzymes (e.g., proteases), this should in fact be possible. So far this approach has not been extended to other types of substrates. 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

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