Encyclopedia of Experiments: Biological Techniques
Ein Abonnement für JoVE ist erforderlich, um diesen Inhalt ansehen zu können. Melden Sie sich an oder starten Sie Ihre kostenlose Testversion.
Transcript
Phosphatidylinositol 4,5-bisphosphate, or PIP2, is a minor membrane-bound phosphorylated phospholipid. Phospholipase C, or PLC, with specific PIP2-binding domains, binds to PIP2, generating secondary messengers essential for several cellular processes.
To detect PLC-PIP2 interactions in vitro, begin with a small unilamellar vesicle, SUV, acidic suspension, comprising PIP2 and phospholipid mixture, one of which is conjugated to a pH-sensitive fluorescent dye.
Pipette the solution into a pre-assembled fabricated microfluidic device's inlet and connect outlet tubing. Transfer and secure the device onto a fluorescence microscope stage. Through gravity, SUVs pass through the microchannel floor, made of a hydrophilic glass coverslip.
The SUVs adsorb on the glass surface, the solution's pH reducing electrostatic repulsion between the two. The SUVs interact with the surface, deforming and fusing to form large vesicles.
Following critical surface coverage, the vesicles rupture spontaneously, forming a supported lipid bilayer, SLB. Pass a buffer through the channel, removing unadsorbed vesicles.
Using a suitable fluorescence channel, image the microchannel to visualize and measure the pH-sensitive dye fluorescence — highly fluorescent at low pH — in the SLB.
Introduce a recombinant PLC solution into the microchannel. The solution's neutral pH, below the PLC's isoelectric point, makes the PLC positively charged.
The recombinant PLC, with its PIP2-binding domain, binds to the PIP2, recruiting negatively-charged hydroxide ions to the bilayer surface. This renders the bilayer surface more basic, shifting the protonation state of the dye and causing fluorescence quenching, indicating PLC-PIP2 interactions.
