Microarrays interaction array

The second type of enzyme application in microfluidics is protein interaction studies. In order to study interactions between enzymes and substrates, peptides, or other proteins, an alternative to microtiter plates is the microarray. These arrays are very often used in conjunction with microfluidic sample and reagent delivery systems alternatively, microchannels can be used to deposit spots in an array. By immobilizing substrates or inhibitors on the array, it is possible to screen large numbers of molecules for their relative abilities to bind a particular enzyme. This is typically accomplished by tagging the enzyme with a fluorescent molecule and using the intensity of the array spots to determine the amount of enzyme bound (Fig. 3a). This is a promising... 

The ultimate goal of microarray-based expression analysis is to acquire a comprehension of the entire cellular process, in order to exploit and to standardize the multidi-menisional relations between genotype and phenotype. However, an increasingly important parameter, which has not yet been substantially taken into account, is the role of cellular translation. This means that mRNA expression data need to be correlated with the assortment of proteins actually present in the cell. One approach is based on the use of microarrays containing double-stranded DNA probes for the analysis of DNA-protein interaction and, thus, the detection and identification of DNA-binding proteins by means of fluorescence [130] or mass spectrometry analysis [131]. Moreover, substantial efforts are currently under way to develop protein, antibody, or even cell arrays, applicable to the cor-... 

Analytical tools have been developed in order to identify carbohydrate structures as well as carbohydrate-binding proteins and to understand their underlying structure-function relationships of protein-carbohydrate and carbohydrate-carbohydrate interactions lectin arrays [16], glycan microarrays [17, 18], glyco-nanoparticles [19], frontal affinity chromatography [20] and carbohydrate tools for metabolic labeling [21]. 

Some arrays used in proteomics contain antibodies covalently bound onto the array surface for immobilization. Then these antibodies capture corresponding antigens from a complex mixture. Afterwards, a series of analysis are carried out. For instance, bound receptors can reveal ligands. With this information in hand, binding domains for protein-protein interactions can be detected. The main problem in using microarray methods for proteomics is that protein molecules must show folding with the array in the correct conformation during the preparation and incubation. Otherwise, protein-protein interactions do not take place. 

We have also carried out other protein-protein interaction assays using p53 protein function microarrays fabricated according to the protocols described above (6). In such assays, we probe the array with a Cy3-labeled solution-phase partner (for example, Cy3-MDM2) in a manner essentially identical to that described above (Subheading 3.5.2.) for the antibody-binding assay. 

Fig. 6. Measurement of the relative amount of ligand bound to each protein in the array. (A) Schematic of on-chip binding assay in which a fluorescently labeled interaction partner binds to the functional, arrayed protein immobilized to the streptavidin-coated surface via the biotinylated BCCP tag. (B) p53 protein function microarray probed with Cy3-labeled GADD45 duplex oligo. Quantification of the signal intensity from each spot allows the effect of polymorphic and functional variation on the DNA binding function of p53 to be determined.

Functional protein microarrays are useful for direct analysis of biochemical activity (e.g., substrate specificity, Refs. 98 and 99), protein protein interaction (100), or small molecule protein interaction. Proteins of interest are over expressed and applied in pure form on addressable arrays, and assays performed (101). 

Protein assays using ordered arrays emerged in the late 90s [13]. Recently Lahiri et al. introduced a technique which enables the fabrication of microarrays for membrane proteins [14]. Printed lipid microspots on y-aminopropylsilane slides have high mechanical stability, independent of whether the lipid is in the gel-or fluid-phase. They immobilized G-protein coupled receptors and studied their interaction with a set of inhibitors. 

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