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  • 標題: 蛍光標識

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The pyridylamination method was originally described in 1978 as a means of analyzing glycan structures with high-sensitivity. Subsequently, the method has been applied to structure analyses of glycans including glycosidase digestion, 2D-mapping by various kinds of HPLC's, partial acetolysis, Smith degradation, methylation analysis, nuclear magnetic resonance, mass spectrometry, and affinity assay for lectins. Glycans on glycoconjugates are liberated by hydrazinolysis followed by N-acetylation. Reducing ends of the released glycans are tagged with 2-aminopyridine by reductive amination. Pyridylamino (PA-) derivatives of glycans with fluorescence and a positive charge have the following advantages: 1. high sensitivity in detection; 2. excellent separation in reversed phase HPLC; 3. high chemical stability under the conditions for structure elucidation; 4. applicable to many authentic methods for glycan analysis.
ポリアクリルアミドゲル電気泳動法による蛍光標識糖分析法 (FACE)
  • 久保見 朋子, 藤田 剛, 松尾 雄志
  • 生物物理化学
  • Vol. 43 (1999) No. 3
  • 公開日: 2009年03月31日
FACE (fluorophore-assisted carbohydrate electrophoresis) is a method for the separation of fluorophore-labeled saccharides by the use of polyacrylamide gel electrophoresis. The method is characterized by high resolution, high sensitivity, and ease of use without expensive giant-sized machine such as mass spectrometry. It has been found to be useful for the analysis of oligosaccharides profiling of N-linked or O-linked glycans after releasing saccharides from glycoproteins and for sequencing oligosaccharides component. Judging from the importance of oligosaccharides in glycoproteins and the changes in oligosaccharides of cancer cell surface, the method described here is hopeful as a unique analysis tool in the field of basic medical science.
DNA profiling has become a standard technique in criminal investigations due to its ability to obtain results from any biological material. PCR-based DNA typing systems have also enabled analyses of DNA obtained from highly decomposed human remains. Based on the length and genomic distribution of the core repeat sequences, two systems are classified as minisatellites and microsatellites (or short tandem repeats). In particular, short tandem repeats (STRs) are abundant classes of polymorphic loci dispersed throughout the entire genome. Each STR locus usually consists of a limited number of the core repeats of 2-5 basepairs, which can easily be resolved by analytical gel electrophoresis. In combination with the use of fluorescence-labelled primers employing different fluorescent dyes, STR loci with overlapping or identical allele size ranges can be analyzed together in the same lane of polyacrylamide gel, thus providing a rapid and accurate method for human identification. Automated DNA profiling by fluorescence-based technology, as well as its application, have been studied and validated for use in forensic laboratory routine.
Fluorescence 2-D difference gel electrophoresis (DIGE) uses spectrally resolvable dyes to label protein samples prior to 2-D electrophoresis. By using different fluorescent dyes to separately label protein samples multiple samples can be co-separated and visualized on a single 2-D gel. Differences between samples are resolved using image analysis software such as DeCyder 2D. This fluorescent multiplexing approach is compatible with mass spectrometry and overcomes many of the disadvantages of traditional 2-D analyses. A broad dynamic range provides more accurate quantitative data than traditional 2-D silver staining techniques while rapid image overlay simplifies image analysis and improves comparative accuracy.