Faye L, Chrispeels M J
Anal Biochem. 1985 Aug 15;149(1):218-24. doi: 10.1016/0003-2697(85)90498-1.
Glycoproteins which bind concanavalin A (Con A) can be located on nitrocellulose sheets after electrophoretic transfer from slab gels, by sequential incubation of the sheets with Con A and peroxidase, and visualization of the peroxidase by an insoluble reaction product. We refer to this method as affinoblotting. Differential elution of Con A from the blots by washing the sheets with different concentrations of alpha-methylglycosides is used to demonstrate the affinity of Con A for the oligosaccharide side chains, and to differentiate between proteins with weak and those with high affinity for Con A. Concanavalin A has a high affinity for the four plant glycoproteins (phaseolin, phytohemagglutinin, jackbean alpha-mannosidase, and the glycosylated precursor of Con A) studied here. Incubation of the blots with alpha-mannosidase and endoglycosidase H (endo H) is used to demonstrate that the oligosaccharide chains can be degraded by glycosidases while the proteins are immobilized on the nitrocellulose. With this approach we show here that the four plant glycoproteins used as models in this study interact with Con A through high-mannose oligosaccharide side chains sensitive to alpha-mannosidase and endo H degradation.
结合伴刀豆球蛋白A（Con A）的糖蛋白，在从平板凝胶进行电泳转移后，可通过依次用Con A和过氧化物酶孵育硝酸纤维素膜，并通过不溶性反应产物使过氧化物酶显色，从而定位在硝酸纤维素膜上。我们将此方法称为亲和印迹法。通过用不同浓度的α-甲基糖苷洗涤膜，从印迹中差异洗脱Con A，用于证明Con A对寡糖侧链的亲和力，并区分对Con A亲和力弱和亲和力高的蛋白质。伴刀豆球蛋白A对这里研究的四种植物糖蛋白（菜豆蛋白、植物血凝素、刀豆α-甘露糖苷酶和Con A的糖基化前体）具有高亲和力。用α-甘露糖苷酶和内切糖苷酶H（内切H）孵育印迹，用于证明在蛋白质固定在硝酸纤维素上时，寡糖链可被糖苷酶降解。通过这种方法，我们在此表明，本研究中用作模型的四种植物糖蛋白通过对α-甘露糖苷酶和内切H降解敏感的高甘露糖寡糖侧链与Con A相互作用。
