[3dem] amount of lipid used for monolayer techinique

[Jing Wang]

Thanks a lot Dianne!
I also explored where the excessive lipid located in the teflon well
by including 10% rhodamin-PE to see the lipid spread by eye. As you
mentioned, lots of lipid aggregated around the rim of the 5mm teflon
well, or it accumulated in the center of the well and sometimes sank
into the buffer reservoir. I have lowered the lipid amount down to1 ul
of 0.2 mM to 0.5 mM mix, it works fine as well. I used mixture of POPC
and POPS, I supposed the mixture should be at fluid phase from 4C to
RT...
Is there special device you used to minimize vibration? I put my
teflon block in a sealed petri-dish on a shelf in the cold room for
the 4C incubation. The shelf seems stable but the cold room is so
noisy with the cooling system goes on all the time. Is that better to
use a fridge instead?
Jing
On Fri, Mar 15, 2013 at 3:09 PM, Dianne Taylor <dianne at bio.fsu.edu> wrote:
> 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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>
>
> 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