extraction of glycan from alcohol insoluble residue

マイクロアレイポリマープロファイリングによる糖鎖の構造と出現性の解明

Glycans are ubiquitous in all domains of life and exhibit unparalleled diversity in structure and function compared to other macromolecules1. However, due to their complexity, variability in biosynthesis and glycosidic linkages, and the paucity of appropriate methods for dissecting glycan structures, our understanding of this diversity in structures and functions is relatively limited2.
Many glycan analysis techniques are destructive and necessitate the breakdown of glycans into their constituent monosaccharides, which can obscure relevant three-dimensional and biological contexts3. Conversely, monoclonal antibodies (mAbs), carbohydrate binding modules (CBMs), lectins, viral agglutinins, and microbial adhesins, known collectively as glycan-recognizing molecular probes (GRMPs)4, recognize and bind to specific epitopes and can be used as tools to detect and discriminate between glycans within complex multi-glycan matrices5,6.
Here, we present microarray polymer profiling (MAPP), a rapid, versatile, and nondestructive method for glycan analysis that is applicable to a broad spectrum of biological samples. The method aims to provide a robust and high-throughput technology for analyzing glycans from diverse biological and industrial/commercial systems. MAPP unites the recognition specificity of glycan-directed molecular probes with reproducible, high-performance microarray screening technology to allow thousands of molecular interactions to be profiled in parallel. The output of this approach is diagnostic insight into the composition and relative abundance of glycans within a sample or tissue of interest.
MAPP can be used as an independent, stand-alone method, or in conjunction with other biochemical techniques, such as immunofluorescence microscopy7,8,9 and monosaccharide or linkage analysis10,11. The technique can also be used to map epitope specificities of novel GRMPs, using arrays printed with pure and structurally well-defined oligosaccharide standards12. A major advantage of MAPP over other methods, such as enzyme-linked immunosorbent assay (ELISA), is its compatibility with small sample volumes13,14. Moreover, MAPP offers significantly higher-throughput analysis15 and provides an effective form of sample preservation, as printed samples are dry and stable when immobilized onto nitrocellulose16.
The binding of GRMPs is generally dependent on the presence of a number of contiguous sugar residues that collectively form a binding site (epitope) that is unique to a particular polysaccharide class (xylan, mannan, xyloglucan, etc.)17. In contrast, the individual sugar residues (xylose, mannose, glucose) that are quantified using most biochemical techniques, for example monosaccharide composition or methylation analysis, can be components of multiple polysaccharide classes and thus difficult to assign18.
MAPP has been developed in response to a technology gap, namely the ability to rapidly analyze multiple glycans from a variety of sources using small amounts of material. MAPP capitalizes on the extensive repertoire of GRMPs that have been developed and characterized over the last three decades12,19,20,21,22,23,24,25,26,27,28,29,30,31,32. The development of MAPP has been an iterative process, with the technique being steadily refined and optimized. There is now a substantial body of literature describing the application of MAPP to various natural and industrial systems where glycans play central roles 5,6,9,10,21,33,34,35,36,37,38,39. Here, we describe the current state of the art for MAPP.

著者スポットライト: MAPP Protocol – Advancing Glycan Analysis 

ハイスループット糖鎖分析のためのマイクロアレイポリマープロファイリング(MAPP)

Watch this Scientific Journal Video about Extraction of Glycan from Alcohol Insoluble Residue at JoVE.com

アルコール不溶性残渣からの糖鎖抽出

まず、機械式TissueLyserを使用して、事前に調製したAIR CDTA混合物を27ヘルツで2分間振とうし、続いて10ヘルツで2時間振とうします。次に、混合物を10, 000 Gで4°Cで10分間遠心分離します。上清を滅菌マイクロ遠心チューブに移します。最後に、水酸化ナトリウムと水素化ホウ素ナトリウムの混合物を残留ペレットに加えます。ペレットを振とうして遠心分離した後、残留ペレットを蒸留水で2〜3回洗浄します。セルラーゼ1ミリグラムあたり30マイクロリットルをペレットに加え、酵素の最適温度でヒートブロック内でチューブを16時間インキュベートします。サンプルを遠心分離し、上清をロータリーシェーカーに保存します。保存したすべての抽出物を、10, 000 G で 4 °C で 10 分間再度遠心分離します。

extraction-of-glycan-from-alcohol-insoluble-residue

Research

JoVE Journal

Biology

Extraction of Glycan from Alcohol Insoluble Residue

81 ビュー. まず、機械式TissueLyserを使用して、事前に調製したAIR CDTA混合物を27ヘルツで2分間振とうし、続いて10ヘルツで2時間振とうします。次に、混合物を10, 000 Gで4°Cで10分間遠心分離します。上清を滅菌マイクロ遠心チューブに移します。最後に、水酸化ナトリウムと水素化ホウ素ナトリウムの混合物を残留ペレットに加えます。ペレットを振とうして遠心分離した後、残...

ビデオ: アルコール不溶性残渣からの糖鎖抽出

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

Microarray polymer profiling (MAPP) is a high-throughput technique for compositional analysis of glycans in biological samples.

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans ...