Production and Application of Glycan Microarrays
Glycans are vital elements of living organisms and are involved in recognition, communication, cell growth and development, motility, and other significant processes. The interactions of glycans with the proteins that bind them provide valuable informatio
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1. Introduction The study of interactions between GBPs and glycan ligands has progressed greatly through the development and use of glycan microarrays. The technology of these arrays allows investigators to observe and analyze GBP interactions on individual chips, each of which can contain hundreds of different glycan structures. Arrays can be used to study a number of GBPs including antibodies specific to tumors or HIV, plant lectins, and virus GBPs such as hemagglutinin (HA) (2). Glycan arrays can be very easily customized to contain desired structures, which gives the capability to explore pathogen-specific glycan interactions (3).
Sridar V. Chittur (ed.), Microarray Methods for Drug Discovery, Methods in Molecular Biology, vol. 632, DOI 10.1007/978-1-60761-663-4_17, © Humana Press, a part of Springer Science+Business Media, LLC 2010
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Busch, McBride, and Head
2. Materials 2.1. Plate Setup
1. Printing Buffer: 300 mM phosphate buffer, pH 8.5 containing 0.005% Tween-20. Store at 4°C long term, but must be at room temperature before use to avoid crystallization. 2. 96-well plates: Nunc 96-well conical bottom (Nunc 249944). 3. 96-well plate lids: Nunc 96-well cap, natural rubber, nonsterile (Nunc 276002). 4. Matrix standard clear 384 well small volume microplates. 5. Matrix universal polystyrene lid use with 96, 384, and 1,536 well plates. 6. USA Scientific Sealing Film (2921-0000). 7. Glycans (natural and/or synthetic) with amine derivatized spacers. Store at −20°C long term, but must be at room temperature when printing.
2.2. Printing
1. SCHOTT Nexterion® Slide H, polymer layer activated with N-Hydroxysuccinimide (NHS) esters which covalently binds amine groups. Store sealed in bags at −20°C long term. The slides must be removed from freezer for at least 4 h (usually overnight) before opening bags and printing. 2. ArrayIt Stealth Micro Spotting Pins (SMP4B and SMP4 models were used and have the same printing quality and produce the same spot size) or acceptable equivalent. Stored in the arrayer head or in manufacturers’ box with tip protection intact.
2.3. Humidification/ Immobilizing
1. Large Pyrex dish (5 cm × 30 cm × 15 cm). 2. Rack, such as test tube. 3. Plastic Wrap.
2.4. Numbering, Blocking, and Storage
1. VWR black lab marker. 2. Slide staining rack, 50 slide rack, and dish (Wheaton 90040). 3. Blocking buffer: 50 mM ethanolamine in 50 mM borate buffer, pH 9.2. Stored at 4°C long term but for best results, should be at room temperature before contacting slides. 4. Glass Pyrex loaf dish with fitted glass lid ( 3 1 4 × 7 7 8 × 4 1 8 ). 5. Glass slide staining rack (Wheaton 900200). 6. Drierite Anhydrous Calcium Sulfate, Indicating (Item#23005). Stored at room temperature in tray on bottom of desiccator
Production and Application of Glycan Microarrays
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boxes. Should be replaced when color indicates-time period varies depending on how often the containers are opened and exposed to humidity. 2.5. Incubation
1. Bioworld PBS 20× solution, pH at 25°C: 7.3–7.5 stored at room temperatur
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