Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(1)

Chemicals and Media

Polyacrylamide-biotin (PAA-biotin) as well as a-L-fucose-polyacryl-amide-biotin (fucose-PAA-biotin) and LewisA-polyacrylamide-biotin (LeA-PAA-biotin) were obtained from Glycotech Corporation (Moscow, Russia). Both biotinylated probes consist of 20 mol% carbohydrate, 5 mol% biotin, and 75 mol% acrylamide, which equals the conjugation of four carbohydrate moieties and one biotin moiety to one polyacrylamide molecule. Neutravidin horseradish peroxidase conjugated (neutravidin-HRP), biotin-hydrazide (EZ-Link Biotin-lethal concentration-hydrazide) and N-hydroxysulfosuccinimide (NHS)-biotin were from Pierce (Rockford, IL).

Percoll and Sephadex (G-50 medium grade) were from Phar-macia (Uppsala, Sweden). Protease inhibitors (Comlate Mini, EDTA-free) were from Roche (Mannheim, Germany). 1,9-Dimethyllene blue (DMMB) (80% pure) was from Aldrich Chem. Co. (Bornem, Belgium). Polystyrene 96-well microtiter plates (maxisorb) were from NUNC (Roskilde, Denmark). Hematoxylin was from J.T. Baker (Chemicals B.V., Deventer, The Netherlands), DePeX mounting medium from BDH Laboratory Supplies (Poole, England).