TechNotes No. 2-1999
Binding
of a plant lectin — horseradish peroxidase conjugate to b–D–galactoside GlycoWell™ plates.
Introduction
Binding of lectins to immobilized saccharides is an efficient
method for evaluting lectin. In addition, competive inhibition lectins1
binding to immobilized saccherides provides a convenient method for screening
and characterization of 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 b–D–galactopyranoside
recognizing lectin, Ricinus communis toxin RCA 1201, to b–D–galactopyranoside
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 a b–D–galactopyranoside
GlycoWell™ plate efficiently binds a b–D–galactopyranoside–recognizing
plant lectin, RCA120 (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-01-006 (b–D–galactopyranoside).
- 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 RCA120-horseradish peroxidase conjugate (SIGMA L2758), 1.0 mL TWEEN/PBS.
- Lectin
solution: 70 µL Lectin stock, 1.75 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 RCA120–horseradish
peroxidase conjugate binds to b–D–galactopyranoside GlycoWell™ plates (SW-01-006),
but not to a GlycoWell™ plate devoid of saccharide (SW-00-001).
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References
1.
Nicolson, G.L. and Blaustein, J. Biochim. Biophys. Acta, 1972, 266,
543.