Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions
Published: June 29, 2023
Abstract
Source: Schoofs, G., et al. A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4. J. Vis. Exp. (2018).
In this video, we describe a flow cytometry-based competitive binding assay to detect the interactions between the CXC Chemokine Receptor 4 (CXCR4) and its fluorescently-labeled natural ligand CXC Chemokine Ligand 12 (CXCL12), in the presence of a CXCR4-targeting small molecule. Incubating CXCR4-expressing cells with lower small molecule concentrations allows CXCR4-CXCL12 binding to some extent, which progressively declines upon a gradual increase in small molecule concentrations.
Protocol
1. Preparation of CXCL12, Assay Buffer, and Jurkat Cells for the Competition Binding Assay Prepare a stock solution of CXCL12AF647 (20 µg/mL; see Table of Materials and Reagents) by dissolving the lyophilized reagent (stored at -80 °C, in the dark) in ultrapure water supplemented with 0.01% (volume/volume) of Polysorbate 20. Store single-use aliquots from this stock solution at -80 °C, protected from light. Prepare assay buffer by adding 40…
Representative Results
Figure 1: Overview of the workflow and illustration of the type of data obtained. (A). The main steps of the protocol are schematized for the negative and positive control samples, and compound-treated samples. Samples without compound pre-incubation and without addition of fluorescently labeled chemokine (CXCL12AF647) are included to determine the background (auto)flu…
Disclosures
The authors have nothing to disclose.
Materials
| BD FACSCanto II | Becton Dickinson | Not applicable | Flow cytometry device |
| BD FACSDIVA Software | |||
| BD FACSArray | Becton Dickinson | Not applicable | Flow cytometry device |
| BD FACSArray System Software | |||
| Rapid flow filter: 0.2 μm aPES | Thermo Scientific | 566-0020 | |
| FlowJo | FlowJo is now a wholly owned subsidiary of BD. | ||
| Vi-CELL | Beckman Coulter | Not applicable | Cell viability analyzer |
| Sigma 3-18 KS | Sigma | Not applicable | Centrifuge |
| AMD3100 | Sigma | A5602-5mg | Specific CXCR4 antagonist |
| h-SDF1a (AF647) | ALMAC | CAF-11-B-01 | Fluorescently-labeled CXCL12, CXCL12AF647 |
| Sterilin microtiter plate, 96-well, U bottom, clear |
Thermo Scientific | 611U96 | |
| Bovine Serum Albumin (BSA) | Sigma | A1933-25G | |
| HBSS (10x), calcium, magnesium, no phenol red | Gibco (Life Technologies) | 14065-049 | |
| HEPES (1M) | Gibco (Life Technologies) | 15630-056 | |
| Dulbecco's Phosphate Buffered Saline (DPBS) | Gibco (Life Technologies) | 14190-094 | |
| Jurkat cells | ATCC | ||
| Reagent reservoir PP | Sigma | BR703411 | |
| Falcon tubes, 50ml | Greiner Bio-One | 227 261 |
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Cite This Article
Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions. J. Vis. Exp. (Pending Publication), e21451, doi: (2023).