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
- 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. 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
- GlycoWell™ plates SW-00-001 (N-acetyl, blank)
and SW-01-004 (a-Neu5Ac).
- 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 Limax flavus-horseradish
peroxidase conjugate (EY Labs H5101), 1.0 mL TWEEN/PBS.
-
Lectin solution: 5 µL Lectin stock, 15.0 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 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).
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