Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany.
Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476, Potsdam, Germany; Institute of Chemistry and Biochemistry, Freie Universität Berlin, Arnimallee 22, 14195, Berlin, Germany.
Carbohydr Res. 2019 Jul 15;481:31-35. doi: 10.1016/j.carres.2019.06.006. Epub 2019 Jun 13.
Interactions of carbohydrates and proteins are essential for many biological processes and glycan microarrays have emerged as powerful tools to rapidly assess these carbohydrate-protein interactions. Diverse platforms to immobilize glycans on glass slides for subsequent probing of the specificities of glycan-binding proteins (GBPs) have evolved. It has been suggested that high local glycan density on microarrays is crucial for detecting low-affinity interactions. To determine the influence of printing efficacy on GBP binding, we compared N-hydroxyl succinimide (NHS)-ester activated glass slides from three different manufacturers and evaluated two different printing buffers. Large differences in binding efficacies of Concanavalin A, peanut agglutinin, and Ricinus communis agglutinin 120 were observed. On some slides, low affinity interactions were missed altogether. Addition of polyethylenglycol (PEG) 400 to the printing buffer significantly enhanced the sensitivity of the binding assays. After monitoring printing efficacy over prolonged printing times, substantial effects resulting from progressing hydrolysis of the NHS-esters during the printing run on one type of slides were found. Printing efficiency of glycans strongly depends on the type of NHS-ester activated slides, the printing buffer, and the printing time. We provide practical advice for selecting the right printing conditions for particular applications.
碳水化合物和蛋白质的相互作用对许多生物过程至关重要，糖芯片已成为快速评估这些碳水化合物-蛋白质相互作用的有力工具。已经开发出多种将糖基固定在玻璃载玻片上的平台，以便随后探测糖结合蛋白（GBP）的特异性。有人认为，糖芯片上的高局部糖密度对于检测低亲和力相互作用至关重要。为了确定打印效果对 GBP 结合的影响，我们比较了来自三个不同制造商的 N-羟基琥珀酰亚胺（NHS）酯激活的玻璃载玻片，并评估了两种不同的打印缓冲液。我们观察到 Concanavalin A、花生凝集素和蓖麻凝集素 120 的结合效率存在很大差异。在一些载玻片上，完全错过了低亲和力相互作用。在打印缓冲液中添加聚乙二醇（PEG）400 显著提高了结合测定的灵敏度。在监测了长时间的打印效果后，发现一种类型的载玻片在打印过程中 NHS 酯水解进展会产生实质性影响。糖的打印效率强烈依赖于 NHS-酯激活载玻片的类型、打印缓冲液和打印时间。我们为特定应用选择正确的打印条件提供了实用建议。
