Protein chips surface functionalization

Fig. 3. Analysis of peptide-lipid interactions using ProteinChip arrays. Vesicles are absorbed to H50 ProteinChip arrays via interactions with C8 functional groups, creating a supported lipid monolayer on the chip surface (A, B). Samples are applied to the chip, incubated for 5 min (C), and washed to remove nonspecifically bound proteins (D). Matrix is added (E), and the array is introduced into the ProteinChip reader, where the laser is fired onto the chip surface. Peptides retained on the surface are finally resolved by TOF-MS, displaying mass-to-charge versus signal intensity (F, G). (Adapted from ProteinChip technology training course, Bio-Rad Laboratories.)...

FIGURE 4.3 The variety of ProteinChip arrays available for sample preparation. (A) The upper arrays represent chemically modified chromatographic surfaces, while the bottom arrays are biochemically modified surfaces. Chemically modified surfaces are used to retain a group of proteins, while biochemically modified surfaces are typically used to isolate a specific protein or functional class of proteins. (B) Protein profile of a cell lysate on different ProteinChip surfaces. As shown in the figure for a selection of protein chips, the individual surfaces retain different groups of proteins, depending on their physiochemical properties. The proteins retained are also dependent on the pH of the sample for the cation and anion exchange surfaces. 

Currently, protein microarrays can be divided into various types depending on the strategies to be chosen. For example, according to the array structure and shape, protein microarrays include 3D-surface structure [30, 31], nanowell [32], and plain chips [33-36], Meanwhile, considering the field of application, protein microarrays can be classified into five categories antibody array, antigen or reserve array, functional array, capture array, and solute array. Table 11.6 shows the differences among them. 

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.

Finally, hydroquinone was connected with its C2 via a pentaethylene glycol Hnker to long alkyl chains with terminal thiol functions. The latter self-assembled on a gold surface to give monolayers. Upon oxidation the hydroquinone was oxidized to quinone and could perform a Diels-Alder reaction with cyclopentadienyle-thanol-conjugated carbohydrate analogs. This set-up is used in chip-based carbohydrate arrays for the evaluation of protein binding and modification [268]. 

---