TechNotes No. 11-2000

Binding of a plant lectin horseradish peroxidase conjugate to b-D-mannoside 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 a a-D-mannopyranoside recognizing lectin, Concanavalin A1, to b-D-mannopyranoside 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. Leave for 15 min. Add 100 mL 1M H2SO4(aq) to each well and read optical density at 490 nm in a microwell plate reader.
Discussion
The results demonstrate that a b-D-mannopyranoside GlycoWell™ plate binds an a-D-galactopyranoside-recognizing plant lectin, Concanavalin A (Figure 1), even though the signal-to-noise ratio is less compared to when a-D-mannopyranoside Glycowell™ plates are used (see TechNote No.1 1999). Thus, GlycoWell plates are suitable for detecting carbohydrate binding properties of proteins.

Materials
Figure 1. A Concanavalin A-horseradish peroxidase conjugate binds to b-D-mannopyranoside GlycoWell™ plates (SW-01-010), but not to a GlycoWell™ plate devoid of saccharide (SW-00-001).

Reference.
1. Reeke, G.N. et al. Annm N.Y. Acad. Sci., 1974,