[3dem] Liposomes in ice

[Aida Razi]

Hi Reza,

We have done so much trouble shooting to get the beautiful cryo-EM liposomes images. Based on our experience, one thing that affect the images, is having sucrose in the buffer. The buffer should be as much as possible close to water, otherwise we saw bubbling effect while we were imaging them.
Also, we put the liposome in holy c-flat grids without any extra layer of the carbon. That reduced the background significantly and we got amazing images of the liposome. However, as liposomes are carbon lover, they got stick to the corner of the holes. Therefore, we played with concentration to get as much as possible of them pushed to the holes and seems 4mg/ml was the best concentration for us.

If you would like, I can send you our liposomes papers with materials and methods on cryo-EM part.

Regards,
Aida Razi/ Dr. Ortega's lab
McGill University
Department of Anatomy and Cell Biology
Montreal, QC.
________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Reza Khayat <rkhayat at ccny.cuny.edu>
Sent: May 12, 2017 10:30:36 AM
To: Ludtke, Steven J; 3dem
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>
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


_______________________________________________
3dem mailing list
3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>
https://urldefense.proofpoint.com/v2/url?u=https-3A__mail.ncmir.ucsd.edu_mailman_listinfo_3dem&d=DwICAg&c=ZQs-KZ8oxEw0p81sqgiaRA&r=GWA2IF6nkq8sZMXHpp1Xpg&m=oA2jpkHqeUTgoaDnmOZhZJ0Ht9Jle6w8wWCE8Ysbx1E&s=Al0k-IvOOSL8k_kowhp3dVXsABnPppq9LeEG7oO9sNo&e=

----------------------------------------------------------------------------
Steven Ludtke, Ph.D.
Professor, Dept of Biochemistry and Mol. Biol.         (www.bcm.edu/biochem<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.bcm.edu_biochem&d=DgMFAg&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=swGrDni_bnbj0SDRv5hphQJtdOkdoQ-0OVRvykOGmk0&s=7k0aceBmiZX_c1AiKvYmgdfvlof36yBIJ3cb4Sh6F1Q&e=>)
Co-Director National Center For Macromolecular Imaging        (ncmi.bcm.edu<https://urldefense.proofpoint.com/v2/url?u=http-3A__ncmi.bcm.edu&d=DgMFAg&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=swGrDni_bnbj0SDRv5hphQJtdOkdoQ-0OVRvykOGmk0&s=vMVLCZ2x3qws1sikvDenN7wwVKbc5CZdCoKuHKU36L8&e=>)
Co-Director CIBR Center                          (www.bcm.edu/research/cibr<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.bcm.edu_research_cibr&d=DgMFAg&c=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo&r=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0&m=swGrDni_bnbj0SDRv5hphQJtdOkdoQ-0OVRvykOGmk0&s=xOaWyEtNdhTND18DZlvEv0ukWg-ciP2Uu7s6uCcAA1Y&e=>)
Baylor College of Medicine
sludtke at bcm.edu<mailto:sludtke at bcm.edu>





-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://mail.ncmir.ucsd.edu/pipermail/3dem/attachments/20170512/25721be7/attachment.html>