[3dem] Re: amount of lipid used for monolayer techinique (Jing Wang)

Amedee des Georges ad2720 at columbia.edu
Mon Mar 4 10:41:42 PST 2013


Hi Jing,

You may want to contact Daniel Levy  <Daniel.Levy at curie.fr> 
He actually has a light microscope specifically designed to look at this. It allows him to closely monitor at which point your mixture switches from mono to multi layers of lipids as you increase the concentration. 

Amedee


-- 
Amedee des Georges
Frank lab
Howard Hughes Medical Institute 
Dept. of Biochemistry and Molecular Biophysics 
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On Mar 2, 2013, at 3:00 PM, 3dem-request at ncmir.ucsd.edu wrote:
> 
>   1. amount of lipid used for monolayer techinique (Jing Wang)
> 
> From: Jing Wang <jing at bu.edu>
> Subject: [3dem] amount of lipid used for monolayer techinique
> Date: March 2, 2013 12:24:42 PM EST
> To: 3dem at ncmir.ucsd.edu
> 
> 
> Dear all,
> I started learning the lipid monolayer technique for protein
> 2D-crystallization 4 months ago and got concern of how much lipid
> should be used. I usually deposit 1 ul of 1 mM lipid on the buffer
> droplet within a teflon well 4mm in diameter and it gave me satisfying
> result based on I can see monolayer faintly contrasting the background
> carbon on the EM grid by negative stain.
> However, by calculation I actually put in 25X excess of lipid required
> to form a monolayer spanning the 4 mm well. I got tough question from
> a faculty member about how do I know I have monolayer transfered to
> the grid. It could be three layers or even more. He suggested that the
> "monolayer" I saw could be extra layer of lipid sitting on top of a
> perfect transferred monolayer attached to the carbon film, so that the
> interesting feature I saw on the grids are not protein, but lipid
> aggregate. From what I read from the literature, people always put
> excess lipid to maintain the monolayer formation. How much excess is
> acceptable in this field? Is there a solid reason for doing that? Is
> there a definite way I can tell whether I have lipid monolayer,
> instead of multi-layer transferred onto the grid? Any comment will be
> greatly appreciate!!!
> Jing
> 
> 
> 
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