Frontal affinity chromatography: sugar-protein interactions.
Frontal affinity chromatography using fluorescence detection (FAC-FD) is a versatile technique for the precise determination of dissociation constants (Kd) between glycan-binding proteins (lectins) and fluorescent-labeled glycans. A series of glycan-containing solutions is applied to a lectin-immobilized column, and the elution profile of each glycan (termed the 'elution front', V) is compared with that (V0) for an appropriate control. Here we describe our standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector. Analysis time for 100 sugar-protein interactions is approximately 10 h, using as little as 2.5 pmol of pyridylaminated (PA) oligosaccharide per analysis. Using FAC-FD, we have so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.
Tateno
Hiroaki
H
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 2, 1-1-1 Umezono, Ibaraki 305-8568, Japan.
Nakamura-Tsuruta
Sachiko
S
Hirabayashi
Jun
J
D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*
D037102:Lectins; D011134:Polysaccharides