TechNotes No. 8-2000

Binding of a plant lectin horseradish peroxidase conjugate to a-sialic acid (a-Neu5Ac) GlycoWell™ plates.

Introduction
Binding of lectins to immobilized surfaces and subsequent inhibition of such a binding event is often used for evaluating lectin inhibitors. The use of GlycoWell™ plates facilitates an ELISA assay involving the binding of lectins to the wells. This TechNote describes the binding of an a-Neu5Ac recognizing lectin, Limax flavus, to a-Neu5Ac GlycoWell™ plates.

Experimental

  1. Lectin incubation. Add 100 L lectin solution to each well. Leave for 45 min
  2. Washing. Empty the wells and wash three times with CovaBuffer. Leave the last wash in the wells for 15 min. Wash with citrate/phosphate buffer once.
  3. Substrate reaction. Add 100 L substrate solution to each well. Read optical density at 490 nm in a microwell plate reader.
Discussion
The results demonstrate that an a-Neu5Ac GlycoWell™ plate efficiently binds an a-Neu5Ac-recognizing plant lectin, Limax flavus (Figure 1). Thus, GlycoWell plates are suitable for detecting carbohydrate binding properties of proteins.

Materials
Figure 1. A Limax flavus-horseradish peroxidase conjugate binds to a-Neu5Ac GlycoWell™ plates (SW-01-004), but not to a GlycoWell™ plate devoid of saccharide (SW-00-001).