A proteolytic method for evaluating O-GlcNAcylation on proteins of similar molecular weight to antibody heavy chain after immunoprecipitation.
IdeZ protease
Immunoprecipitation
O-GlcNAc
Post-translational protein modification
Journal
Analytical biochemistry
ISSN: 1096-0309
Titre abrégé: Anal Biochem
Pays: United States
ID NLM: 0370535
Informations de publication
Date de publication:
15 12 2020
15 12 2020
Historique:
received:
23
05
2020
revised:
12
10
2020
accepted:
21
10
2020
pubmed:
2
11
2020
medline:
27
5
2021
entrez:
1
11
2020
Statut:
ppublish
Résumé
Investigating a protein of interest that runs at the same molecular weight as antibody heavy chain is a frequent deterrent to its evaluation by immunoprecipitation. Methods of minimizing the detection of the immunoprecipitating antibody are available. However, these still present a barrier to evaluating if intracellular proteins are modified by the O-GlcNAc post-translation protein modification due to interfering glycosylation on antibodies. IdeZ protease specifically cleaves antibody at the hinge region, allowing collapse of the antibody fragments to 25 kDa after denaturation. Thus, this proteolytic method uniquely allows evaluation of O-GlcNAcylation of proteins of interest formerly obscured by antibody heavy chain.
Identifiants
pubmed: 33129762
pii: S0003-2697(20)30533-9
doi: 10.1016/j.ab.2020.114001
pmc: PMC7718295
mid: NIHMS1643797
pii:
doi:
Substances chimiques
Immunoglobulin Heavy Chains
0
Peptide Hydrolases
EC 3.4.-
Acetylglucosamine
V956696549
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
114001Subventions
Organisme : NIGMS NIH HHS
ID : P30 GM122731
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK091277
Pays : United States
Organisme : NIDDK NIH HHS
ID : R01 DK100595
Pays : United States
Informations de copyright
Copyright © 2020 Elsevier Inc. All rights reserved.
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