Assays For Measuring Content Mixing
Mixing of aqueous contents from vesicles as a result of lysis, fusion or physiological permeability can be detected fluorometrically using low molecular weight soluble tracers.
1. Fluorescence quenching assays with ANTS/DPX: ANTS is a polyanionic fluorophore, while DPX is a cationic quencher. The assay is based on the collisional quenching of them. Separate vesicle populations are loaded with ANTS or DPX, respectively. When content mixing happens, ANTS and DPX collide and fluorescence of ANTS monitored at 530 nm, with excitation at 360 nm is quenched. This method is performed at acidic pH and high concentration.
2. Fluorescence enhancement assays with Tb3+/DPA: This method is based on the fact that chelate of Tb3+/DPA is 10,000 times more fluorescent than Tb3+ alone. In the Tb3+/DPA assay, separate vesicle populations are loaded with TbCl3 or DPA. The formation of Tb3+/DPA chelate can be used to indicate vesicle fusion. This method is good for protein free membranes.
3. Single molecule DNA assay. A DNA hairpin composed of 5 base pair stem and poly-thymidine loop that is labeled with a donor (Cy3) and an acceptor (Cy5) at the ends of the stem was encapsulated in the v-SNARE vesicle. We separately encapsulated multiple unlabeled poly-adenosine DNA strands in the t-SNARE vesicle. If the two vesicles, both ~100 nm in diameter, dock and a large enough fusion pore forms between them, the two DNA molecules should hybridize, opening up the stem region of the hairpin and switching the Förster resonance energy transfer (FRET) efficiency (E) between Cy3 and Cy5 from a high to a low value.
Read more about this topic: Lipid Bilayer Fusion, Assays To Measure Membrane Fusion
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