β-Lactamase-Based Conductimetric Biosensor Assay for Protein-Protein Interactions
Published: May 31, 2023
Abstract
Source: Vandevenne, M., et al. The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions. J. Vis. Exp. (2018)
This video demonstrates a conductimetric biosensor assay to study the interaction of an antibody with a target antigen immobilized on a transducer chip. The biosensor is engineered to have a bifunctional chimeric protein — a β-lactamase enzyme with a functional catalytic site fused to a nanobody for binding to the immobilized target antigen. Upon binding, the catalytic hydrolysis of an antibiotic substrate by the β-lactamase enzyme generates an electrical signal — confirming the antigen-antibody interaction.
Protocol
1. Protein Sample Preparation Produce and purify the hybrid protein BlaP-cAb-Lys3 as reported in our previous study. Store the protein in 50 mM phosphate buffer pH 7.4 with the following composition: 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 dissolved in 800 mL of distilled water Fix the pH of the solution to 7.4 before adjusting the final volume of the solution to 1 L. Filter sterilize the protein solution. Prepare a hen egg …
Divulgaciones
The authors have nothing to disclose.
Materials
| Reagents | |||
| KH2PO4 | Sigma-Aldrich | V000225 | |
| K2HPO4 | Sigma-Aldrich | 1551128 | |
| NaCl | Sigma-Aldrich | S7653 | |
| Tris–HCl | Roche | 10812846001 | |
| EDTA | Sigma-Aldrich | E9884 | |
| KCl | Sigma-Aldrich | P9541 | |
| Na2HPO4 | Sigma-Aldrich | NIST2186II | |
| 2-mercaptoethanol | Sigma-Aldrich | M6250 | |
| Alanine | Sigma-Aldrich | A7627 | |
| HClO4 | Fluka | 34288 | 1M HClO4 solution, distributor : Sigma-Aldrich |
| Casein hydrolysate | Sigma-Aldrich | 22090 | |
| Benzylpenicillin sodium | Sigma-Aldrich | B0900000 | |
| Hen egg white lysozyme | Roche | 10837059001 | |
| Heptane | Sigma-Aldrich | 246654 | |
| Methanol | Sigma-Aldrich | 322415 | |
| Ammonium hydroxide solution | Sigma-Aldrich | 380539 | 28% NH3 in H2O, purified by double-distillation (concentrated?) |
| Laboratory consumables | |||
| 6-well plate | Greiner Bio-One | 657165 | CELLSTAR 6-Well Plate |
| Equipment | |||
| pH meter | WTW | 1AA110 | Lab pH meter inoLab pH 7110 |
| Vacuum and filtration system | Nalgene | NALG300-4100 | Filter holders with receiver, distributor : VWR |
| Potentiometric sensor chips | manufactured by Yunus and colleagues (ref 16) | ||
| PGSTAT30 Autolab | Metrohm Autolab | discontinued, successor Autolab PGSTAT302N | |
| Digital multimeter, METRAHit 22M | Gossen Metrawatt | discontinued, successor Metrahit Base |
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Citar este artículo
β-Lactamase-Based Conductimetric Biosensor Assay for Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21402, doi: (2023).