Glycan arrays consisting of 367 diverse glycans with and without the presence of one of three spacers (sp2, sp3 or sp4 [49 (link)]) were prepared from two previously described glycan libraries [83 (link),84 (link)]. Amine containing glycans with spacer’s sp2, sp3 or sp4 were synthesised as previously described [49 (link)] and glycans without spacers were amine functionalised as previously published [85 (link)]. All glycans were suspended in 1:1 DMF:DMSO at a concentration of 500 mM and were printed onto SuperEpoxy 2 glass slides (ArrayIt, Sunnyvale, CA) using a ArrayIt SpotBot Extreme array spotter in a six pin subarray print per glass slide format. All glycans were printed in replicates of four, including four FITC control spots as well as additional position controls (S1 Fig ), per subarray using SMP4 pins and a contact time of 1 second at 60% relative humidity, with pins being reloaded after every 12 spots.
Prior to performing glycan array experiments, slides were scanned using a ProScanArray Microarray 4-laser scanner (Perkin Elmer, Waltham, MA) using the blue argon 488 laser set to the FITC settings (492 nm excitation and 517 nm emission). Array slides were blocked with 0.1% BSA in 50 mM phosphate buffered saline (PBS), pH 7.4 for 5 min at 22°C. After washing with PBS, each slide was dried by placing them in an empty 50 mL tube and centrifuging for 5 min at 200 x g (900 rpm). Recombinant TconTS-LD (2 μg) was incubated at a molar ratio of 1:2:3 with anti His-tag mouse polyclonal antibody (10 mg/mL, Cell Signalling Technology), anti mouse-IgG-Alexa555 conjugated rabbit polyclonal antibody (2 mg/mL, Life Technologies) and anti rabbit-IgG-Alexa555 conjugated goat polyclonal antibody (2 mg/mL, Life Technologies) in 50 mM PBS, pH 7.4 containing 0.1% BSA and 10 mM maltotriose for 15 min on ice protected from light. All subarrays on the slide were isolated using a Gene Frame (1.5 x 1.6 cm, 65 μL, Abgene, Epsom, UK) prior to the addition of the TconTS-LD-antibody mix to the array. A coverslip was applied to the GeneFrame and array slides incubated for 30 min at 22°C in the dark. The GeneFrame and coverslip were subsequently removed and the slide gently washed twice with 50 mM PBS, pH 7.4 containing 0.01% TWEEN 20 and 10 mM maltose, and once with 50 mM PBS, pH 7.4 containing 10 mM maltose. Slides were dried by centrifugation for 5 min at 200 x g (900 rpm), allowed to air dried for a further 5 min, and the fluorescence associated with the array spots detected using the microarray scanner settings outlined above. Image analysis and spot visualisation was performed using the ProScanArray software, ScanArray Express (Perkin Elmer). The resulting images were visually examined. Fluorescence signals were judged as being positive, if all four replicates for a glycan were clearly detectable (S1 Fig ).
Prior to performing glycan array experiments, slides were scanned using a ProScanArray Microarray 4-laser scanner (Perkin Elmer, Waltham, MA) using the blue argon 488 laser set to the FITC settings (492 nm excitation and 517 nm emission). Array slides were blocked with 0.1% BSA in 50 mM phosphate buffered saline (PBS), pH 7.4 for 5 min at 22°C. After washing with PBS, each slide was dried by placing them in an empty 50 mL tube and centrifuging for 5 min at 200 x g (900 rpm). Recombinant TconTS-LD (2 μg) was incubated at a molar ratio of 1:2:3 with anti His-tag mouse polyclonal antibody (10 mg/mL, Cell Signalling Technology), anti mouse-IgG-Alexa555 conjugated rabbit polyclonal antibody (2 mg/mL, Life Technologies) and anti rabbit-IgG-Alexa555 conjugated goat polyclonal antibody (2 mg/mL, Life Technologies) in 50 mM PBS, pH 7.4 containing 0.1% BSA and 10 mM maltotriose for 15 min on ice protected from light. All subarrays on the slide were isolated using a Gene Frame (1.5 x 1.6 cm, 65 μL, Abgene, Epsom, UK) prior to the addition of the TconTS-LD-antibody mix to the array. A coverslip was applied to the GeneFrame and array slides incubated for 30 min at 22°C in the dark. The GeneFrame and coverslip were subsequently removed and the slide gently washed twice with 50 mM PBS, pH 7.4 containing 0.01% TWEEN 20 and 10 mM maltose, and once with 50 mM PBS, pH 7.4 containing 10 mM maltose. Slides were dried by centrifugation for 5 min at 200 x g (900 rpm), allowed to air dried for a further 5 min, and the fluorescence associated with the array spots detected using the microarray scanner settings outlined above. Image analysis and spot visualisation was performed using the ProScanArray software, ScanArray Express (Perkin Elmer). The resulting images were visually examined. Fluorescence signals were judged as being positive, if all four replicates for a glycan were clearly detectable (
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