Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities
Abstract
:1. Introduction
2. Material and Methods
2.1. Plasmids
2.2. Cell Lines, Transfection Method, and Reagents
2.3. Subtractive Phage Display Panning for Isolating RHO-GTP Specific hs2dAb
2.4. Hs2dAb Purification
2.5. RHO GTPases Purification
2.6. ELISA Assays
2.7. Loading Recombinant Proteins with GTPγS/GDP
2.8. Immunofluorescence Staining
2.9. Endogenous RHO Proteins Intracellular Antibodies Co-Immunoprecipitation Assays
2.10. GST-RBD Assay
2.11. Affinity Measurement
2.12. BRET
2.12.1. Molecular Cloning
2.12.2. BRET2 Titration Curves and Stimulation Assays
2.12.3. BRET2 Measurements
3. Results
3.1. Phage Display Selection of a New High Affinity GTP-Bound RHO Conformational hs2dAb
3.2. Characteriation of the RH57 Intracellular Antibody Binding Properties
3.3. Evaluating the RH57 intracellular antibody as a biosensor of RHOA-GTP.
4. Discussion
5. Patents
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Keller, L.; Bery, N.; Tardy, C.; Ligat, L.; Favre, G.; Rabbitts, T.H.; Olichon, A. Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities. Antibodies 2019, 8, 8. https://doi.org/10.3390/antib8010008
Keller L, Bery N, Tardy C, Ligat L, Favre G, Rabbitts TH, Olichon A. Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities. Antibodies. 2019; 8(1):8. https://doi.org/10.3390/antib8010008
Chicago/Turabian StyleKeller, Laura, Nicolas Bery, Claudine Tardy, Laetitia Ligat, Gilles Favre, Terence H. Rabbitts, and Aurélien Olichon. 2019. "Selection and Characterization of a Nanobody Biosensor of GTP-Bound RHO Activities" Antibodies 8, no. 1: 8. https://doi.org/10.3390/antib8010008