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<div>Hi Reza, </div>
<div>We routinely work with (mostly) unilamellar vesicles that are ~100 nm in diameter with lipid concentration varying from 1- 10 mg/ml. Your 200 nm vesicles may need thicker ice to be prepared well. We have had success with various lipid composition. We prepare
vesicles by reconstitution using BioBeads to remove detergents and extrude them through 100 nm filters. The vesicle size follows a normal distribution, usually varying from 80 to 120 nm. It appears that you extruded your vesicles from lipids hydrated with
a buffer solution, which in our hands tended to generate multi. Did you check if DOPG alone can make good vesicles? After extrusion did negative stain EM find multilamellar vesicles? When you ran your vesicles (loaded to the bottom) through a density gradient
(e.g. 5%, 20% and 35% sucrose), were they able to float to the 5% layer and appeared translucent? Did you use lower salt (~50 mM) to help vesicles separated from each other by charge repulsion? Other details we cared include: 1) 10% water added right before
freezing to help maintain the vesicle shape; 2) glow-discharge with amylamine; 3) load vesicles twice and then blot. 4) a small amount of trehalose to help them stay. We wrote a step-by-step protocol for Kv channel reconstitution (<span class="jrnl" title="Journal of visualized experiments : JoVE">J
Vis Exp</span>. 2013 Jul 13;(77):e50436), which might be helpful to your case. </div>
<div>Hope these help. Best, Qiu-Xing</div>
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<span style="font-weight:bold">From: </span>3dem <<a href="mailto:3dem-bounces@ncmir.ucsd.edu">3dem-bounces@ncmir.ucsd.edu</a>> on behalf of Reza Khayat <<a href="mailto:rkhayat@ccny.cuny.edu">rkhayat@ccny.cuny.edu</a>><br>
<span style="font-weight:bold">Date: </span>Friday, May 12, 2017 at 10:30 AM<br>
<span style="font-weight:bold">To: </span>Steven Ludtke <<a href="mailto:sludtke@bcm.edu">sludtke@bcm.edu</a>>, "<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>" <<a href="mailto:3dem@ncmir.ucsd.edu">3dem@ncmir.ucsd.edu</a>><br>
<span style="font-weight:bold">Subject: </span>Re: [3dem] Liposomes in ice<br>
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<p>Hi,</p>
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</p>
<p>We are preparing extruded liposomes (200nm). Problem is we're getting very few liposomes in ice. The starting lipid concentration (DOPG:
<a href="https://avantilipids.com/product/840475/">https://avantilipids.com/product/840475/</a>) is 4mg/ml.</p>
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<p>Reza<br>
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Reza Khayat, PhD
<div>Assistant Professor </div>
<div>City College of New York</div>
<div>Department of Chemistry</div>
<div>New York, NY 10031</div>
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<div id="divRplyFwdMsg" dir="ltr"><font style="font-size:11pt" color="#000000" face="Calibri,sans-serif"><b>From:</b> Ludtke, Steven J <<a href="mailto:sludtke@bcm.edu">sludtke@bcm.edu</a>><br>
<b>Sent:</b> Friday, May 12, 2017 10:27 AM<br>
<b>To:</b> Reza Khayat<br>
<b>Subject:</b> Re: [3dem] Liposomes in ice</font>
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<div>You'll have to define exactly what you mean by liposomes. Extruded SUVs are one of our favorite test/training specimens because they are so easy to prepare and freeze. When most people say liposome, they mean something much larger. You'll need to give
some details if you want useful advice. What are the lipid constituents, how large are the liposomes, how are they prepared, etc. Would also be useful to see a representative image or two.
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<div class="">On May 12, 2017, at 9:18 AM, Reza Khayat <<a href="mailto:rkhayat@ccny.cuny.edu" class="">rkhayat@ccny.cuny.edu</a>> wrote:</div>
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Hi,</div>
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We're trying to visualize liposomes in ice but are having some serious difficulty. Can anyone suggest some tricks that may be helpful? Thanks.</div>
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Best wishes,</div>
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Reza<br class="">
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Reza Khayat, PhD
<div class="">Assistant Professor </div>
<div class="">City College of New York</div>
<div class="">Department of Chemistry</div>
<div class="">New York, NY 10031</div>
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