Practical considerations for printing high-density glycan microarrays to study weak carbohydrate-protein interactions.

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. Image 1 • Optimization of printing conditions afforded high density glycan microarrays. • NHS-Activated slides from different manufacturers hydrolize with different rates during printing runs. • Slides from different manufacturers provided different signal intensities after interrogation with glycan-binding proteins. • Glycan microarray printing has to be carefully fine-tuned depending on the particular application. [ABSTRACT FROM AUTHOR]