Optimized Microscale Protein Aggregation Suppression Assay: A Method for Evaluating the Holdase Activity of Chaperones.
Aggregation suppression
DnaK
Holdase
Malate dehydrogenase
SYPRO Orange
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
medline:
7
8
2023
pubmed:
4
8
2023
entrez:
4
8
2023
Statut:
ppublish
Résumé
Many molecular chaperones act as holdases by binding hydrophobic regions of substrates to prevent aggregation. Therefore, measuring holdase activity is an amenable method to determine chaperone activity. The holdase function is reliably and easily achieved by monitoring the suppression of heat-induced aggregation of well-characterized model protein substrates. However, the standard assay format requires large amounts of protein and hence is not applicable to all proteins. Using DnaK from Escherichia coli and heat-induced aggregation of malate dehydrogenase, we describe a protocol for absorbance and fluorescence-based miniaturized versions of the standard aggregation suppression assay that are affordable and have wide application for low abundance holdases. The assay can be used for both fundamental characterization of holdase function in proteins and screening of inhibitors of holdase activity.
Identifiants
pubmed: 37540431
doi: 10.1007/978-1-0716-3342-7_10
doi:
Substances chimiques
Protein Aggregates
0
Escherichia coli Proteins
0
Molecular Chaperones
0
HSP70 Heat-Shock Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
113-123Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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