Microarray surface preparation

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

Peptide microarrays are prepared by immobilizing many peptide molecules on the surface of a solid support in a small area in an addressable fashion. The immobilization can be achieved via in situ synthesis or chemical ligation through a covalent bond. A hydrophihc linker between the sohd surface and the peptide usually is added to minimize steric hinderance caused by the sohd support. The most commonly used solid support for microarray printing is a standard microscope glass slide. Other solid supports also have been used such as polystyrene, nitrocellulose membranes, PVDF membranes, Hybond ECL membranes, gold surfaces, and chemical vapor deposited diamond films. 

Next, in order to demonstrate the efficacy of this cell surface engineering approach, cell microarrays were prepared using a micropattemed poly(2-methacryloyloxyethyl phosphorylcholine [MPC]) (PMPC) polymer brush surface as the substrate. PMPC brushes were prepared on a silicon (Si) wafer via surface-initiated atom transfer radical polymerization, as previously described elsewhere [35]. Selective decomposition of the initiators immobilized on the Si wafer using UV light irradiation resulted in micropatterning of the... 

Fig. 14. A biphasic plot to determine and for the oxidation of Vivid Red by CYP3A4 immobilised on a microarray surface. The microarray-generated kinetic data set for the turnover of Vivid Red by a CYP3A4-BCCP P0R-BCCP complex (Fig. 13) was fitted to a biphasic, multlsite model using non-linear regression.Tlie resultant catalytic parameters, and describe the enzymatic activity when only a single molecule ot substrate is bound in the active site at any one time these values conform well with literature values obtained with baculosome preparations ot CYP3A4.

There are many protocols and different types of platforms available, e.g. GeneChips from Affymetrix, Illumina Bead Arrays, Arrays from Agilent, Applied Biosystems, GE Healthcare, customized spotted cDNA microarrays etc. the basic procedure for a large-scale measurement of gene expression involves the preparation of total or mRNA from the biological sample(s) under investigation (e.g. candidate tissue) and the hybridization of copied labelled RNA or cDNA to the DNA elements on the array surface (Fig. 1). 

Slides specifically selected for microarray applications should be used. They are available as ultracleaned (an important consideration) and untreated for those who wish to prepare their own surfaces or they can be purchased with a variety of precoated surface chemistries (e.g., lysine, aldehyde, or epoxide). The densities of reactive groups and surface coating uniformity are difficult to control. Thus, if lot-to-lot slide consistency is most important factor, consider using commercially available slides that are quality controlled. 

Others such as Macas et al. (1998) successfully adapted the Biomek 2000 (Beckman Coulter), a commonly used liquid handling robot, to prepare microarray slides using a specially constructed print head and quill pins. Up to 28 microscope slides could be placed on a work surface for printing. Biomek s HDRT head was adapted to accept microarray quill pins held between two parallel plates with holes drilled on 9-mm centers to dip into 96-well source plates. The quill pins were spring-loaded similar to the design... 

Finally, an understanding of the making of a microarray is fundamentally important to those interested in producing "spotted" arrays and properly using them. While complementary (cDNA) microarray fabricahon on glass slides has been well studied, we have less experience with the attachment of oligonucleotides and the preparation of protein arrays. Moreover, additional substrates and surface chemistries that may be better suited for printing proteins are now available. 

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. 

Sugars having maleimide groups linked to the anomeric position underwent selective reaction with maleimides tethered on surfaces for the preparation of sugar microarrays.115... 

Oligonucleotide and peptide microarrays can be prepared in situ with light-directed synthesis on a glass surface in conjunction with either a photolithographic method or using a micromirror device (2,11-15). However, these methods are not useful for most organic synthesis. Furthermore, such approaches require equipment that is not readily available. Spot synthesis on cellulose membrane is another in situ synthesis method, but the resulting microarrays are low density (16). 

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