Protein microarray analysis

VanMeter AJ, Rodriguez AS, Bowman ED et al (2008) Laser capture microdissection and protein microarray analysis of human non-smaU cell lung cancer differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type. Mol Cell Proteomics 7 1902-1924... 

Kader, H.A., et al. (2005) Protein microarray analysis of disease activity in pediatric inflammatory bowel disease demonstrates elevated serum PLGF, IL-7, TGF-betal, and IL-12p40 levels in Crohn s disease and ulcerative colitis patients in remission versus active disease. Am J Gastroenterol. 100, 414-23. 

Lue, R. Y, Zhu, Q., Li, D. (2004). Site-specific peptide immobilization strategies for the rapid detection of kinase activity on microarrays. Site-specific immobilization of biotinylated proteins for protein microarray analysis. Enzymatic profiling system in a small-molecule microarray. Intein-mediated biotinylation of proteins and its application in a protein microarray. Developing site-specific immobilization strategies of peptides in a microarray. Methods Mol. Biol. 278, 191-204. 

In functional protein microarrays, set of proteins of interest or an entire proteome is over expressed, purified and spotted in an addressable microarray surface. The most obvious target molecules that can effectively be used to investigate protein binding and therefore protein microarray analysis are monoclonal antibodies or IgG. However, unlike DNA microarrays, antibody arrays may pose some practical concerns ... 

Microarray detection methods In the early days of protein microarray analysis, mostly radioisotope-based labeling was applied, especially for phosphorylated proteins [99]. Later, stable isotope-coded amino acids (SILAC) proved useful in in vivo incorporation to cell cultures [100]. Both methods were very sensitive, requiring only minute sample amounts to reveal protein expression differences [101]. The use of an enhanced chemiluminescence (ECL) technique using X-ray film exposure or a phosphor imager instrument was. 

Rogers, P. D. et al., Differential expression of genes encoding immunomodulatory proteins in response to amphotericin B in human mononuclear cells identified by cDNA microarray analysis, J. Antimicrob. Chemother., 50, 811, 2002. 

Figure 1.13 Complexity reduction for improved proteomic analysis using protein microarrays.

In this chapter, we will survey the kinds of solid supports (substrates) and surface chemistries currently used in the creation of nucleic acid and protein microarrays. Which are the best supports and methods of attachment for nucleic acids or proteins Does it make sense to use the same attachment chemistry or substrate format for these biomolecules In order to begin to understand these kinds of questions, it is important to briefly review how such biomolecules were attached in the past to other solid supports such as affinity chromatography media, membranes, and enzyme-linked immxm-osorbent assay (ELISA) microtiter plates. However, the microarray substrate does not share certain unique properties and metrics with its predecessors. Principal among these are printing, spot morphology, and image analysis they are the subjects of subsequent chapters. 

This chapter will review some of the key applications presented by protein microarrays. The use of profein microarrays sfems from works on gene expression arrays described earlier. However, rmlike ifs predecessors whose process formats (mutation detection, polymorphism screening, gene expression analysis, etc.) are essentially based upon solid-phase hybridization of nucleic acid complementary strands, the protein array may play different roles and comprise a variety of formafs. 

How do we then envision the protein microarray as a proteomics tool We now estimate the human genome to comprise around 30,000 genes. For gene expression analysis using DNA microarrays, 1000 to 10,000 gene elements are often used. Since proteins undergo posttranslational modification (>200 different types see McDonald and Yates, 2000, Reference 40) and can occur as isoforms and multiprotein complexes, the number of protein expression elements needs to be much larger. 

Pawlak, M., Schick, E., Bopp, M.A., Schneider, M.J., Oroszlan, P., and Ehrat, M., Zeptosen s protein microarrays a novel high performance microarray platform for low abundance protein analysis, Proteomics, 2, 383-393, 2002. 

Sheehan KM, Calvert VS, Kay EW et al (2005) Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma. Mol Cell Proteomics 4 346-355... 

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