The Consortium for Functional Glycomics (http://www.functionalglycomics.org/ ) provided glycan microarrays (v4.2) prepared as described previously 45 (link),47 (link). For galectin recognition of glycans on the printed glycan microarray, slides were incubated with 0.2 µM or 5 µM Gal-4, or 5 µM Gal-8 in TSM binding buffer + 14 mM 2-ME for 1h at room temperature in a dark humid chamber. The slide was washed by successive immersion in TSM containing 0.05% Tween 20 (4 times) and TSM (4 times). The slide was incubated with Alexa Fluor-488-streptavidin. After 1h at room temperature in a dark humid chamber, we washed the slide by successive immersion in TSM containing 0.05% Tween 20 (4 times), TSM (4 times), and water (4 times). The slide was dried by microcentrifugation and an image of bound fluorescence was obtained using a microarray scanner (Scan Array Express, PerkinElmer Lifer Sciences). Integrated spot intensities were determined using Imagene software (BioDiscovery).
Protocol detail
Glycan Microarray Binding Assay
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Alexa fluor 488
Buffer
Fluorescence
Gal 8
Galectin
Glycan
Immersion
Microarray
Scan
Streptavidin
Tween 20
Corresponding Organization :
Other organizations : Emory University, Scripps Research Institute, University of Bern, N.D. Zelinsky Institute of Organic Chemistry
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Variable analysis
independent variables
- Concentration of Gal-4 (0.2 µM or 5 µM)
- Concentration of Gal-8 (5 µM)
- Binding of Gal-4 and Gal-8 to glycans on the printed glycan microarray
- Integrated spot intensities of bound fluorescence
- TSM binding buffer
- 2-ME concentration (14 mM)
- Incubation time (1 hour)
- Incubation temperature (room temperature)
- Washing steps (4 times in TSM containing 0.05% Tween 20, 4 times in TSM, 4 times in water)
- Alexa Fluor-488-streptavidin labeling
- Microarray scanner (Scan Array Express, PerkinElmer Lifer Sciences)
- Imagene software (BioDiscovery) for integrated spot intensity determination
- Not explicitly mentioned
- Not explicitly mentioned
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