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).

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