Next Article in Journal
Tracking Single Cells Motility on Different Substrates
Next Article in Special Issue
Fibrin Clot Formation and Lysis in Plasma
Previous Article in Journal / Special Issue
IsoSearch: An Untargeted and Unbiased Metabolite and Lipid Isotopomer Tracing Strategy from HR-LC-MS/MS Datasets
Open AccessProtocol

Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties

1
School of Biological Sciences, IQB3, University of Edinburgh, The King’s Buildings, Max Born Crescent, CH Waddington Building, Edinburgh, Scotland EH9 3BF, UK
2
School of Biological Sciences and Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, The King’s Buildings, Edinburgh EH9 3BF, UK
*
Author to whom correspondence should be addressed.
Both authors contributed equally to this work.
Methods Protoc. 2020, 3(3), 55; https://doi.org/10.3390/mps3030055
Received: 29 June 2020 / Revised: 14 July 2020 / Accepted: 21 July 2020 / Published: 30 July 2020
(This article belongs to the Special Issue Feature Issue on the Development of Novel Cutting Edge Protocols)
Quantitative microdialysis is a traditional biophysical affinity determination technique. In the development of the detailed experimental protocol presented, we used commercially available equipment, rapid equilibrium dialysis (RED) devices (ThermoFisher Scientific), which means that it is open to most laboratories. The target protein and test compound are incubated in a chamber partitioned to allow only small molecules to transition to a larger reservoir chamber, then reversed-phase high performance liquid chromatography (RP-HPLC) or liquid chromatography–mass spectrometry (LC–MS) is used to determine the abundance of compound in each chamber. A higher compound concentration measured in the chamber that contains the target protein indicates binding. As a novel, and differentiating contribution, we present a protocol for mathematical analysis of experimental data. We provide the equations and the software to yield dissociation constants for the test compound-target protein complex up to 0.5 mM KD, and we quantitatively discuss the limitations of affinities in relation to measured compound concentrations. View Full-Text
Keywords: label free screening; affinity determination; dialysis; KD determination; promiscuity; aggregation; nonspecific binding; protein binding analysis; chromatography; drug discovery label free screening; affinity determination; dialysis; KD determination; promiscuity; aggregation; nonspecific binding; protein binding analysis; chromatography; drug discovery
Show Figures

Graphical abstract

MDPI and ACS Style

Shave, S.; Pham, N.T.; Śmieja, C.B.; Auer, M. Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties. Methods Protoc. 2020, 3, 55. https://doi.org/10.3390/mps3030055

AMA Style

Shave S, Pham NT, Śmieja CB, Auer M. Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties. Methods and Protocols. 2020; 3(3):55. https://doi.org/10.3390/mps3030055

Chicago/Turabian Style

Shave, Steven; Pham, Nhan T.; Śmieja, Connor B.; Auer, Manfred. 2020. "Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties" Methods Protoc. 3, no. 3: 55. https://doi.org/10.3390/mps3030055

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop