Reliable and sensitive detection of glycosaminoglycan chains with immunoblots.
chondroitin sulfate proteoglycan
glycosaminoglycans
heparan sulfate proteoglycan
immunoblot
western blot
Journal
Glycobiology
ISSN: 1460-2423
Titre abrégé: Glycobiology
Pays: England
ID NLM: 9104124
Informations de publication
Date de publication:
09 02 2021
09 02 2021
Historique:
received:
17
04
2020
revised:
24
06
2020
accepted:
25
06
2020
pubmed:
3
7
2020
medline:
22
2
2022
entrez:
3
7
2020
Statut:
ppublish
Résumé
Complex glycans play vital roles in many biological processes, ranging from intracellular signaling and organ development to tumor growth. Glycan expression is routinely assessed by the application of glycan-specific antibodies to cells and tissues. However, glycan-specific antibodies quite often show a large number of bands on immunoblots and it is hard to interpret the data when reliable controls are lacking. This limits the scope of glycobiology studies and poses challenges for replication. We sought to resolve this issue by developing a novel strategy that utilizes an immunoreaction enhancing technology to vastly improve the speed and quality of glycan-based immunoblots. As a representative case study, we used chondroitin sulfate glycosaminoglycan (CS-GAG) chains as the carbohydrate target and a monoclonal antibody, CS-56, as the probe. We discovered that preincubation of the antibody with its antigenic CS-GAG chain distinguishes true-positive signals from false-positive ones. We successfully applied this strategy to 10E4, a monoclonal anti heparan sulfate GAGs (HS-GAGs) antibody, where true-positive signals were confirmed by chemical HS-GAG depolymerization on the membrane. This evidence that glycan-specific antibodies can generate clear and convincing data on immunoblot with highly replicable results opens new opportunities for many facets of life science research in glycobiology.
Identifiants
pubmed: 32614944
pii: 5866559
doi: 10.1093/glycob/cwaa060
pmc: PMC7874388
doi:
Substances chimiques
Antibodies, Monoclonal
0
chondroitin sulfate glycosaminoglycan
0
Chondroitin Sulfates
9007-28-7
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
116-125Subventions
Organisme : NIGMS NIH HHS
ID : T32 GM007200
Pays : United States
Informations de copyright
Published by Oxford University Press 2020.
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