Detection of lectins using ligand blotting and polyacrylamide-type glycoconjugate probes.
A sensitive and convenient method for detection of the carbohydrate-binding activity of lectins was established using the combination of blotting of lectins on polyvinylidene difluoride membranes, carbohydrate-conjugated biotinylated polyacrylamide-type probes (carbohydrate-bp probes), horseradish peroxidase-streptavidin, and detection by enhanced chemiluminescence of the enzyme reaction. This method was tested for detection of four plant lectins blotted on the membrane: concanavalin A was detectable down to 100 ng by mannose-bp probe, Ricinus communis agglutinin 120 to as low as 5 ng by N-acetyllactosamine-bp probe, soybean agglutinin to 1 microgram by beta-N-acetyl-D-galactosamine-bp probe, and wheat germ agglutinin to 5 ng by beta-N-acetyl-D-glucosamine-bp probe. All four lectins were detectable on an electroblotted membrane after SDS-polyacrylamide gel electrophoresis. This method was used to detect recombinant human galectin-3 in Escherichia coli cell lysates and mannan-binding protein in human serum. These results indicate that this method is widely applicable to convenient detection and characterization of lectins in crude samples.
Kamemura
K
K
Kato Cytoprotein Network Project, ERATO, JST, Kanagawa, Japan.
Kato
S
S
D000180:Acrylic Resins / Q000737:chemistry*; D000328:Adult; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D037502:Galectin 3; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006001:Glycoconjugates / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D037241:Mannose-Binding Lectins; D015335:Molecular Probes; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000943:Antigens, Differentiation; D002352:Carrier Proteins; D017931:DNA Primers; D037502:Galectin 3; D005982:Glutathione Transferase; D006001:Glycoconjugates; D008024:Ligands; D037241:Mannose-Binding Lectins; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D000099166:Polyacrylamides