In this video, we demonstrate protein-phosphoinositide interactions in a microfluidic pattern using the pH modulation assay, using pH-sensitive fluorescent dye tagged to phosphatidylethanolamine lipid of a supported lipid bilayer, called a microfluidic platform. The modulations in pH after protein binding to phospholipid cause fluorescence quenching.
Protocol
1. Forming Supported Lipid Bilayers (SLBs) Transfer 100 µL of PI(4,5)P2-containing SUVs into a 0.65 mL microcentrifuge tube. Adjust the pH of the solution to ~3.2 by adding 6.4 µL of 0.2 N hydrochloric acid. NOTE: Confirm the pH of the solution with a pH meter equipped with a micro pH probe (see table of materials). Pipette 10 µL of the pH-adjusted SUV solution into each channel through the inlet and apply pressure through the …
Representative Results
Figure 1: Assessing the integrity of SLBs and microchannels. (A) An image illustrating high-quality SLBs and microchannels. (B) Incomplete fusion. (C) Fused microchannels. (D) Dust particle trapped within a microchannel. (E) Air bubbles trapped within microchannels.