TechNotes No. 9-2000

Binding of a plant lectin — horseradish peroxidase conjugate to 2–acetamido–2–deoxy–b–D–galactopyranoside 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 2–acetamido–2–deoxy–b–D–galactopyranoside recognizing lectin, Ricinus communis toxin RCA 120¹, to 2–acetamido–2–deoxy–b–D–galacto-pyranoside GlycoWell™ plates.

Experimental

1. Lectin incubation. Add 100 µL 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 µL substrate solution to each well. Leave for 15 min. Add 100 mL 1M H₂SO_4(aq) to each well and read optical density at 490 nm in a microwell plate reader.

• GlycoWell™ plates SW-00-001 (N-acetyl, blank) and SW-01-002 (2–acetamido–2–deoxy–b–D–galacto-pyranoside).
• PBS buffer pH 7.2, 0.15 M Na⁺: 24.0 g NaCl, 0.6 g KCl, 3.46 g Na₂HPO₄·2H₂O, 0.6 g KH₂PO₄, 3 L H₂O.
• TWEEN/PBS: 0.10 mL TWEEN 20, 200 mL PBS.
• CovaBuffer: 116.9 g NaCl, 10.0 g MgSO₄·7H₂O, 0.50 mL TWEEN 20, 1 L PBS.
• Citrate-Phosphate buffer, pH 5.0: 7.30 g citric acid, 11.86 g Na₂HPO₄·2H₂O, 1 L H₂O.
• Lectin stock: 1.0 mg RCA 120-horseradish peroxidase conjugate (SIGMA L4401), 1.0 mL TWEEN/PBS.
• Lectin solution: 70 µL Lectin stock, 0.7 mL TWEEN/PBS, filtered through a 0.22 micron filter.
• Substrate solution: 6.0 mg o-phenylenediamine, 10.0 mL citrate/phosphate buffer, 5 µL H₂O₂, freshly prepared.

Figure 1. A RCA 120-horseradish peroxidase conjugate binds to 2–acetamido–2–deoxy–b–D–galacto-pyranoside GlycoWell™ plates (SW-01-002), but not to a GlycoWell™ plate devoid of saccharide (SW-00-001).