Investigating Protein Binding with the Isothermal Ligand‐induced Resolubilization Assay
Abstract: Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand‐induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand‐induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate‐linked moiety X motif 5 (NUDT5), poly [ADP‐ribose] polymerase 1 (PARP1), and protein arginine N‐methyltransferase 1 (PRMT1). Ligand‐induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand‐induced protein solubility were assessed by Coomassie blue staining for SDS‐PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput.
- Standort
-
Deutsche Nationalbibliothek Frankfurt am Main
- Umfang
-
Online-Ressource
- Sprache
-
Englisch
- Erschienen in
-
Investigating Protein Binding with the Isothermal Ligand‐induced Resolubilization Assay ; day:12 ; month:02 ; year:2024 ; extent:10
ChemBioChem ; (12.02.2024) (gesamt 10)
- Urheber
-
Prout‐Holm, Riley A.
van Walstijn, Cerissa C.
Hitsman, Alana
Rowley, Michael J.
Olsen, Jonas E.
Page, Brent D. G.
Frankel, Adam
- DOI
-
10.1002/cbic.202300773
- URN
-
urn:nbn:de:101:1-2024021314142269871269
- Rechteinformation
-
Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
- Letzte Aktualisierung
-
15.08.2025, 07:26 MESZ
Datenpartner
Deutsche Nationalbibliothek. Bei Fragen zum Objekt wenden Sie sich bitte an den Datenpartner.
Beteiligte
- Prout‐Holm, Riley A.
- van Walstijn, Cerissa C.
- Hitsman, Alana
- Rowley, Michael J.
- Olsen, Jonas E.
- Page, Brent D. G.
- Frankel, Adam
Ähnliche Objekte (12)
An enhanced isothermal amplification assay for viral detection
The role of water in protein-ligand binding : a comprehensive study by crystallography and isothermal titration calorimetry
A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor
Development of Quantitative Rapid Isothermal Amplification Assay for Leishmania donovani
Simultaneous Quantification of Oestrogen and Progesterone Receptors by a Ligand Binding Assay in Frozen Sections
A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)
Predicting target-ligand interactions using protein ligand-binding site and ligand substructures
Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions
Selection of a Ligand-Binding Neutralizing Antibody Assay for Benralizumab: Comparison with an Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Cell-Based Assay
Investigating the ligand binding mode of two peptide-activated G protein-coupled receptrors : Y2R and CMKLR1
Derangement model of ligand-receptor binding
Conformational Changes in Ligand Binding Processes
An enhanced isothermal amplification assay for viral detection
The role of water in protein-ligand binding : a comprehensive study by crystallography and isothermal titration calorimetry
A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor
Development of Quantitative Rapid Isothermal Amplification Assay for Leishmania donovani
Simultaneous Quantification of Oestrogen and Progesterone Receptors by a Ligand Binding Assay in Frozen Sections
A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)
Predicting target-ligand interactions using protein ligand-binding site and ligand substructures
Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions
Selection of a Ligand-Binding Neutralizing Antibody Assay for Benralizumab: Comparison with an Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Cell-Based Assay
Investigating the ligand binding mode of two peptide-activated G protein-coupled receptrors : Y2R and CMKLR1
Derangement model of ligand-receptor binding
Conformational Changes in Ligand Binding Processes
An enhanced isothermal amplification assay for viral detection
The role of water in protein-ligand binding : a comprehensive study by crystallography and isothermal titration calorimetry
A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor
Development of Quantitative Rapid Isothermal Amplification Assay for Leishmania donovani
Simultaneous Quantification of Oestrogen and Progesterone Receptors by a Ligand Binding Assay in Frozen Sections
A competitive ligand-binding assay for the detection of neutralizing antibodies against dostarlimab (TSR-042)
Predicting target-ligand interactions using protein ligand-binding site and ligand substructures
Isothermal chemical denaturation assay for monitoring protein stability and inhibitor interactions
Selection of a Ligand-Binding Neutralizing Antibody Assay for Benralizumab: Comparison with an Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Cell-Based Assay
Investigating the ligand binding mode of two peptide-activated G protein-coupled receptrors : Y2R and CMKLR1
Derangement model of ligand-receptor binding