Protein microarrays preparation

The functional microarray typically consists of a collection of full-length functional proteins or protein domains printed on glass slides that are then exposed to a protein preparation from a cell that represents the entire proteome of that cell. This method is useful in determining protein-protein interactions. In addition, this method is useful in predicting the interaction of proteins with DNA, RNA, phospholipids, and small molecules. RPA includes glass slides on which a cellular protein preparation is fixed and then probed with a known antibody. This method helps in identifying the proteins that are altered and cannot bind with a known antibody in the proteome of diseased cell types. This method also identifies the proteins that are altered as a result of phosphorylation or other posttranslational modifications in normal and disease conditions or under growth conditions. 

And, how is it that membranes cast upon glass substrates are now used to prepare protein microarrays We will address these questions in good time. 

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. 

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. 

Lee Y, Lee EK, Cho YW, et al. (2003) Pro-teoChip a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies. Proteomics 3, 2289-304. 

Preparing protein microarrays by soft-landing of mass-selected ions. Science 1351. ... 

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. 

Arenkov et al. prepared poly(acrylamide) gel pads for use in protein microarrays [199], The gels were prepared by photopolymerization of acrylamide and crosslinkers. Capture probes were immobilized, either by use of glutaraldehyde or by converting some of the acrylamide groups into hydrazides and subsequent coupling of aldehyde-modified antibodies to the pending hydrazide groups. Then, immunoassays were performed on the pads, either assays with directly labeled analytes or sandwich assays. Furthermore, the gel pads were used for enzyme activity studies. 

O Brien, J., Stickney, J. T, Porter, M. D. (2000). Preparation and characterisation of self-assemhled douhle-stranded DNA (dsDNA) microarrays for protein ... 

Recently, a small molecule fluorophore phosphosensor technology referred as Pro-Q Diamond dye has been developed to detect and quantitate phosphorylated amino acids within peptides and proteins in microarrays. ° In addition to binding assays, fluorescence detection methods have also been developed for functional assays. For example, microarrays of quenched fluorescent substrates can be used to detect protease or esterase activities in the analytes. In this method, quenched fluorescent substrates are prepared by coupling the peptide substrate to coumarin, a fluorescent dye. These peptide substrates are then spotted onto the solid support... 

Houseman et al. prepared a peptide chip by Diels-Alder-mediated reaction of kinase-peptide substrates with a self-assembled monolayer of alkanethiolates on gold surface. Peptide phosphorylation was determined by incubating the peptide microarrays with c-src protein-tyrosine kinase followed by quantitation with a phos-phoryl imager. In the presence of soluble inhibitors at a range of concentrations, dose-dependent inhibition of phosphorylation against a number of peptide substrates could be determined on a single chip. ... 

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