Dissipative Microgravimetry Technique to Study Protein-Lipid Bilayer Interaction
Published: June 29, 2023
Abstract
Source: Matos, A. L. L., et al. Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator. J. Vis. Exp. (2018).
This video demonstrates a dissipative microgravimetry technique to investigate the binding of proteins to lipid bilayers. Small unilamellar vesicles are added to coat the surface of the quartz sensor of a microbalance. Utilizing the calcium ion-dependent binding of the target phospholipid-binding protein to the bilayer, changes in the oscillation frequency of the sensor and the dissipation of the oscillation are monitored to determine the nature of the protein-lipid bilayer binding.
Protocol
NOTE: Buffers should be filtered using a 0.22-µm filter and degassed by a vacuum for 1 h. 1. Lipid Vesicle Preparation Use 2-mL glass tubes. Dissolve each lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesterol (Chol), in a mixture of chloroform/methanol (50:50 v/v) to prepare a clear 5 mM lipid solution. Dissolve…
Representative Results
Table 1: Lipid composition and formation data of the SLB Composition ΔΔF/Hz after formation of SLBs ΔΔD*10-6 after formation of SLB POPC/POPS(80 : 20) 26.3 ± 0.2 0.26 ± 0.03 POPC/PI(4,5)P2(95 : 5) 26.5 …
Divulgations
The authors have nothing to disclose.
Materials
| Chemicals | |||
| Calcium chloride | Merck | 017-013-00-2 | 99% |
| Chloroform | Roth | 4432.1 | 99% |
| DOPC | Avanti | 850375P | |
| EGTA | PanReac AppliChem | A0878 | 99% |
| HEPES | PanReac AppliChem | A1069 | |
| Methanol | PanReac AppliChem | A3493 | |
| PiP2 | Avanti | 850155P | |
| POPC | Avanti | 850457P | |
| POPS | Avanti | 840034P | |
| Sodium chloride | PanReac AppliChem | A1149 | |
| SDS | Roth | 183 | |
| Trisodium citrate | PanReac AppliChem | A3901 | |
| Equipment | |||
| Extruder Liposofast | Avestin | ||
| Qsense E4 Analyzer | Qsense | ||
| QSense Dfind | Qsense | ||
| Pump IPC 4 | Ismatec | ISM 930 | |
| QSX 303 SiO2 Silicon dioxide 50nm | Qsense | QSX 303 | |
| PC Membranes 0.05μm | Avanti polar lipids | 610003 | |
| OriginPro | OriginLab Corporation | Version 8 and 9 |
Tags
Citer Cet Article
Dissipative Microgravimetry Technique to Study Protein-Lipid Bilayer Interaction. J. Vis. Exp. (Pending Publication), e21443, doi: (2023).