[3dem] amount of lipid used for monolayer techinique

[Dianne Taylor]

Hello,

An excess of lipid is always necessary to produce a monolayer of good 
integrity, possibly the excess creates compression to pack the lipid 
molecules more tightly together. I make stocks at 1 mg/ml for 
convenience and dilute to 0.5 mg/ml for application (1 ul for a 5 mm 
well). Your amount is a bit high, it could be reduced some. You don't 
say which lipids are being used, if they are fluid or gel phase, but 
my values work for either.

In my experience (25 years) the above lipid concentration will be a 
monolayer. The excess appears to go to the side of the well and drops 
to the bottom as the solvent evaporates.

There are, however; many things that can disrupt the monolayer and 
create folds, vesicles, and other lipid structures in your EM sample. 
These can definitely make evaluating samples harder. Mechanical 
disruption is the biggest problem. Vibrations from surroundings can 
break up the monolayer so it's important to find a stable and 
vibration free place to work. Contact with carbon film also causes 
problems. Monolayers lift best at an air/water interface as in 
reticulated carbon film. If you are seeing patches on carbon film 
this may be the remnants of the monolayer.

Hope this helps,
Dianne Taylor


At 01:24 PM 3/2/2013, Jing Wang wrote:
>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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>3dem at ncmir.ucsd.edu
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Dianne W. Taylor
Associate in Research
Institute of Molecular Biophysics
Florida State University
Tallahassee, FL 32306-4380
Phone: (850) 644-4104
E-mail:  dianne at bio.fsu.edu