ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells
Published: June 29, 2023
Abstract
Source: Elias, S. et al., A BW Reporter System for Studying Receptor-Ligand Interactions. J. Vis. Exp. (2019)
This video demonstrates the reporter assay-based detection of the receptor-ligand interaction. The successful receptor-ligand interaction activates the downstream signaling pathway, causing the generation and release of cytokines as secretory reporter molecules in the solution, which is confirmed using ELISA.
Protocol
1. Generation of a Plasmid that Expresses the Chimeric Construct NOTE: The aim is to generate a plasmid that expresses the extracellular domain of the receptor of interest fused to the transmembrane and intracellular domains of the mouse CD3ζ chain (Figure 1A). Retrieve the sequence of the extracellular domain of the receptor of interest including the signal peptide. Obtain DNA material that is expected to express this recepto…
Representative Results
Figure 1: The structure of the chimeric receptor-CD3ζ and the design of PCR primers. (A) General structure of an extracellular receptor fused to the transmembrane and intracellular domains of mouse CD3ζ. (B) The extracellular domain of the receptor was amplified with a 3' primer which also included nucleotides of CD3ζ. (C) The intracellular and transmembrane domains of CD3ζ…
Divulgaciones
The authors have nothing to disclose.
Materials
| AccuPrep, Plasmid Mini Extraction Kit | Bioneer | K-3030 | |
| Anti-mouse IL-2 | BioLegend | 503702 | |
| Biotin anti-mouse IL-2 | BioLegend | 503804 | |
| ELISA plates | De-groot | 60-655061 | |
| Fetal Bovine Serum | Sigma | F7524-500ml | |
| G418 | Mercury | MBS3458105GM | |
| Gene Pulser II | Bio-Rad | 165-2105, 165-2106, 165-2107, 165-2108, 165-2109, 165-2110 | |
| L-Glutamine | Biological industry | 03-020-1B | |
| MEM Non-Essential Amino Acids Solution | Biological industry | 01-340-1B | |
| Penicillin-Streptomycin Solution | Biological industry | 03-031-1B | |
| Peroxidase streptavidin | Jackson ImmunoResearch | 016-030-084 | |
| PureLink HiPure Plasmid Filter Maxiprep Kit | ThermoFisher Scientific | K210016 | |
| RPMI-1640 Medium | Biological industry | 30-2001 | |
| Sodium Pyruvate | Biological industry | 03-042-1B | |
| TMB | SouthernBiothech | 0410-01 |
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Citar este artículo
ELISA-Based Reporter Assay to Study the Receptor-Ligand Interaction in BW Cells. J. Vis. Exp. (Pending Publication), e21452, doi: (2023).