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
- Lectin incubation. Add 100 µL lectin
solution to each well. Leave for 45 min
- 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.
- Substrate
reaction. Add 100 µL 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
- GlycoWell™ plates SW-00-001 (N-acetyl, blank)
and SW-01-010 (b-D-mannopyranoside).
- 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 Concanavalin
A -horseradish peroxidase conjugate (SIGMA L6397), 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 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,