2.6. Galectin Binding Assay with Immobilized Glycans and Neo-Glycoproteins

The synthesized glycans and neo-glycoproteins were analyzed for binding of Gal-3 and Gal-1 in 96-well microtiter plate formats [37,41]. Glycans 11 and 12, as well as the corresponding LacNAc-LacNAc-linker-NH₂ 1a and LacdiNAc-LacNAc-linker-NH₂ 2a (deprotected forms of 1 and 2) were immobilized via the amino group in aminoreactive microtiter plates (Immobilizer Amino, Nunc, Wiesbaden, Germany). The immobilization of 5 nmol glycan in sodium carbonate buffer (100 mM, pH 9.6) was done overnight. For immobilizing neo-glycoproteins, 5 pmol protein were incubated in PBS (pH 7.5) in MaxiSorp microtiter plates (Nunc) overnight. Wells were then washed with PBS-Tween (0.05% (v/v)) and blocked with 2% BSA in PBS followed by incubation for one hour with galectins diluted in EPBS. Incubation with anti-His₆-peroxidase (Roche) was done subsequently. Microtiter plates were read out at 495 nm after conversion of OPD substrate (o-phenylenediamine, Dako, Hamburg, Germany). Measured data were analyzed using Sigma Plot (Systat software GmbH, Erkrath, Germany).