Техника называется<strong> C</strong> Omprehensive<strong> M</strong> Icroarray<strong> P</strong> Olymer профилирования (CoMPP) для характеристики растений гликанов клеточной стенки описано. Этот метод сочетает в себе специфичность моноклональных антител, направленных на определенный гликана-эпитопы с миниатюрной микрочипов аналитическая платформа, позволяющая показ гликана вхождения в широкий спектр биологических контекстах.
Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 & 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.
CoMPP это быстрый и чувствительный метод для профилирования гликана составе сотни растительного происхождения образцов в течение нескольких дней. Этот метод дополняет уже имеющиеся бактерий и млекопитающих гликана платформ массив для высокопроизводительного скрининга углеводов взаи…
The authors have nothing to disclose.
IEM хотел бы отметить датского исследовательского совета (FTP и FNU) для финансирования. ERL признает, поддержка ARC DP гранта. AB благодарит за поддержку АРК центра передового опыта в завод грант клеточных стенок.
Name of the reagent | Company | Catalogue number | Comments (optional) |
3 mm Tungsten Carbide beads | Qiagen | 69997 | |
Collection microtubes (1.2 mm) | Qiagen | 19560 | 1.5 ml microfuge tubes can also be used |
Qiagen TissueLyser II | Qiagen | 85300 | |
3 mm glass beads | Sigma Aldrich | Z143928 | |
CDTA | Sigma Aldrich | 34588 | |
Cadmium oxide | Sigma Aldrich | 202894 | |
1,2-diaminoethane | Sigma Aldrich | 03550 | |
Nitrocellulose membrane (0.22 μm pore size) | GE-water & process technologies | EP2HY00010 | different pore sized membranes are suitable for different pin types |
Xact II microarrayer robot | Labnext | 001A | the Xact II robot was fitted with a custom 20 x 20 cm ceramic plate to which the nitrocellulose membrane is attached |
Xtend RM microarray pins | Labnext | 0037-350 | pins must be suitable for spotting on membranes |
384 well microtiter plates (polypropylene) | Greiner | 781207 | |
Anti-glycan monoclonal antibodies | Plant Probes/ CarboSource/Biosupplies |
Websites; PlantProbes (www.plantprobes.net), Carbosource (www.carbosource.net) and Biosupplies (www.biosupplies.com.au). | |
Anti-Rat IgG (whole molecule) – Peroxidase antibody produced in goat. | Sigma | A9037 | the type of secondary antibodies depends on the primary antibody used (e.g. raised in rat, mouse, goat etc). |
SIGMAFAST 3,3′-Diaminobenzidine tablets | Sigma | D4293 | the type of developing reagent depends on the secondary antibodies used and the detection method (colourmetric, or chemiluminecent). |
SuperSignal West Pico Chemiluminescent Substrate | Thermoscientific | 34080 | see above |
Xplore Image Processing Software | LabNext | 008 | many software types with automatic gridding tools are available to measure pixel value of microarray spots. |
Plant polysaccharides | Sigma/Megazyme |