[3dem] amount of lipid used for monolayer techinique

[Jing Wang]

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