Protein-surface interactions fluorescence-labeling 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.

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

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