Model Neural Membrane Droplet Interface Bilayers from Brain Total Lipid Extract for Studying Membrane-Peptide Interactio
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stock solution preparation. Alm stock and final solution preparation is described elsewhere.21 Fresh aliquots of Aβ are used for all tests, with aliquots prepared as follows: dissolve lyophilized stock Aβ to 0.5 mg/mL in acetonitrile (Sigma), freeze at -80°C, lyophilize, re-dissolve in 2.5 M NaOH to 1 mg/mL and distribute into aliquots of 10-20 μL (10-20 μg each), re-freeze, and relyophilize. When ready for testing, fresh Aβ stock solution is prepared by dissolving the lyophilized Aβ in aqueous buffer to a concentration of 0.5 mg/mL, and the stock is diluted with buffer or liposomes to achieve final concentrations as indicated herein. Circular Dichroism. CD spectroscopy is performed using 1-mm path length quartz cells with a Jasco J-810 CD spectropolarimeter. Scan parameters and details of spectra analysis (background correction and drift removal) are described elsewhere.21 Pure Aβ samples consist of 40 μg/mL Aβ. Aβ-liposome samples include 160 μg/mL lipid and 40 μg/mL Aβ. Cuvette and sample temperature are maintained using a Jasco temperature controller (JWJTC-484) with at least 5 minutes allowed for temperature equilibration with each new sample and after any temperature change. To study secondary structure over the course of several weeks, samples are saved after testing and stored at 4°C in a refrigerator or at 37°C in a temperature-controlled hot room. DISCUSSION Differential Scanning Calorimetry and DIB Formation Initial tests are performed to understand if DIB formation is possible using total lipid extracted from the brain. Recent studies showed that heating to 50°C is required to form monolayers when using total lipid extracted from E. coli (eTLE).21 Heating is required because the lipids undergo a thermotropic gel-fluid transition between 35-50°C. We thus perform DSC measurements with BTLE liposomes to determine if the mixture undergoes a phase transition near room temperature. DSC is also performed on liposomes made from dipalmitoyl phosphatidylcholine (DPPC) which is known to undergo an endothermic transition between 41-43°C. Thermograms from DSC scans between 20-80°C are shown in Figure 1A. DPPC undergoes a transition with a peak around 42°C as expected, while BTLE liposomes display no such endothermic transition. Assuming BTLE is in the fluid phase across the range of temperature tested, DSC results suggest that DIB formation should be possible without the need for heating above the transition temperature (Tg) as required for eTLE, DPPC, and even DMPC (Tg = 25°C).21 Based on the fact that BTLE lipids Figure 1. A) Differential Scanning Calorimetry (DSC) of buffer, DPPC liposomes in buffer, and BTLE liposomes in buffer. DPPC did not show a phase transition at shows an endothermic transition (Tg) around 42°C while BTLE temperatures above RT, trials to form shows no clear transition. B) States of DIB formation using the DIBs with BTLE are initially RAM. Typical droplet size is between 300-700nL with food coloring performed at room temperature while added to assist droplet visualization. C) Bilayer formatio
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