Advait Thakore

G-protein coupled receptors(GPCRs) are cell transmembrane protein receptors that initiate a range of signal pathways in cells, which may take form as a phosphorylation cascade, cAMP signaling, or calcium ion signaling to elicit a cellular response. One common GPCR experiment uses Giant Plasma Membrane Vesicles (GPMVs) which consist of a GPCR linked to an acceptor and donor Forster resonance energy transfer (FRET) biosensor. GPMV protocol is standard and while the GPMV procedure has been used successfully in yielding GPMVs for past experiments, there is still room for optimization in the GPMV preparation procedure to output a greater yield of GPMVs by altering incubation temperature, incubation times, and reagent concentration. A control GPMV preparation following standard protocol was performed along with multiple experimental GPMV preparations for testing each variable. GPMV yield was analyzed via spectrofluorometer data and compared with the control to determine the effect of each condition on GPMV yield and functionality. It was found that increased temperature and incubation time displayed the largest increase in GPMV yield from the standard protocol, while NEM reagent concentration did not display any clear tends in GPMV yield from the standard. Increased temperature and time of incubation produced an additive effect on GPMV yield. This experiment resulted in optimization of the GPMV protocol in producing GPMVs via incubation temperature and time. This will benefit labs that utilize GPMVs, as there will be more GPMVs available from the GPMV protocol for further GPCR experiments.