[3dem] Liposomes in ice

Jiang,Qiu-Xing qxjiang at ufl.edu
Fri May 12 08:14:51 PDT 2017


Hi Reza,
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 (J Vis Exp. 2013 Jul 13;(77):e50436), which might be helpful to your case.
Hope these help. Best, Qiu-Xing
-

From: 3dem <3dem-bounces at ncmir.ucsd.edu<mailto:3dem-bounces at ncmir.ucsd.edu>> on behalf of Reza Khayat <rkhayat at ccny.cuny.edu<mailto:rkhayat at ccny.cuny.edu>>
Date: Friday, May 12, 2017 at 10:30 AM
To: Steven Ludtke <sludtke at bcm.edu<mailto:sludtke at bcm.edu>>, "3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>" <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>>
Subject: Re: [3dem] Liposomes in ice


Hi,


We are preparing extruded liposomes (200nm). Problem is we're getting very few liposomes in ice. The starting lipid concentration (DOPG: https://avantilipids.com/product/840475/) is 4mg/ml.


Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031
________________________________
From: Ludtke, Steven J <sludtke at bcm.edu<mailto:sludtke at bcm.edu>>
Sent: Friday, May 12, 2017 10:27 AM
To: Reza Khayat
Subject: Re: [3dem] Liposomes in ice

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.

On May 12, 2017, at 9:18 AM, Reza Khayat <rkhayat at ccny.cuny.edu<mailto:rkhayat at ccny.cuny.edu>> wrote:

Hi,

We're trying to visualize liposomes in ice but are having some serious difficulty. Can anyone suggest some tricks that may be helpful? Thanks.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031


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