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A practical protocol for carbohydrate microarrays.

ABSTRACT: We have established a high-throughput biochip platform for constructing carbohydrate microarrays. Using this technology, carbohydrate-containing macromolecules of diverse structures, including polysaccharides, natural glycoconjugates, and mono- and oligosaccharides coupled to carrier molecules, can be stably immobilized on a glass chip without chemical modification. Here, we describe a practical protocol for this technology. We hope that anyone who has access to a standard cDNA microarray facility will be able to explore this technology for his or her own research interest. We also provide an example to illustrate that the carbohydrate microarray is also a discovery tool; this is particularly useful for identifying immunologic sugar moieties, including complex carbohydrates of cancer cells and sugar signatures of previously unrecognized microbial pathogens.

SUBMITTER: Wang R

PROVIDER: S-EPMC7120908 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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A practical protocol for carbohydrate microarrays.

Wang Ruobing R Liu Shaoyi S Shah Dhaval D Wang Denong D

Methods in molecular biology (Clifton, N.J.) 20050101

We have established a high-throughput biochip platform for constructing carbohydrate microarrays. Using this technology, carbohydrate-containing macromolecules of diverse structures, including polysaccharides, natural glycoconjugates, and mono- and oligosaccharides coupled to carrier molecules, can be stably immobilized on a glass chip without chemical modification. Here, we describe a practical protocol for this technology. We hope that anyone who has access to a standard cDNA microarray facili ...[more]

PMID: 16350957

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Project description:The transport and catabolic machinery involved in carbohydrate utilization by Lactobacillus acidophilus was characterized genetically by using whole-genome cDNA microarrays. Global transcriptional profiles were determined for growth on glucose, fructose, sucrose, lactose, galactose, trehalose, raffinose, and fructooligosaccharides. Hybridizations were carried out by using a round-robin design, and microarray data were analyzed with a two-stage mixed model ANOVA. Differentially expressed genes were visualized by hierarchical clustering, volcano plots, and contour plots. Overall, only 63 genes (3% of the genome) showed a >4-fold induction. Specifically, transporters of the phosphoenolpyruvate:sugar transferase system were identified for uptake of glucose, fructose, sucrose, and trehalose, whereas ATP-binding cassette transporters were identified for uptake of raffinose and fructooligosaccharides. A member of the LacS subfamily of galactoside-pentose hexuronide translocators was identified for uptake of galactose and lactose. Saccharolytic enzymes likely involved in the metabolism of monosaccharides, disaccharides, and polysaccharides into substrates of glycolysis were also found, including enzymatic machinery of the Leloir pathway. The transcriptome appeared to be regulated by carbon catabolite repression. Although substrate-specific carbohydrate transporters and hydrolases were regulated at the transcriptional level, genes encoding regulatory proteins CcpA, Hpr, HprK/P, and EI were consistently highly expressed. Genes central to glycolysis were among the most highly expressed in the genome. Collectively, microarray data revealed that coordinated and regulated transcription of genes involved in sugar uptake and metabolism is based on the specific carbohydrate provided. L. acidophilus's adaptability to environmental conditions likely contributes to its competitive ability for limited carbohydrate sources available in the human gastrointestinal tract.

Dataset Information

A practical protocol for carbohydrate microarrays.

Publications

A practical protocol for carbohydrate microarrays.

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