[3dem] Alkylamine vapor while glow discharge

Sacha De Carlo sachadecarlo at yahoo.com
Fri Feb 6 04:39:25 PST 2009


Hello,

poly-amines are used to pre-treat carbon to adsorb DNA/protein complexes. I don't have a lot of experience with it, but I happened to work with people like A. Stasiak who looked at RecA and the like.

If you do cryo-EM of single particles, and your particles love to stick to the carbon instead of spreading nicely in the holes, it is useful to treat the carbon so that it becomes hydrophobic. True, spreading of the droplet will not be as nice and you may end up depositing a larger droplet and using more samples. In my case, having looked at several RNA Polymerases from many species, a pretreatment of the grid with choloroform and no glow-discharge has proven to be very useful.

Polylysine can be used to affect the 'orientation' of particles in the thin vitreous ice layer. Again I remember we used to get top-views of a bacterial pore-forming toxin that would show only side-views otherwise. 

Cheers,
Sacha



================================================
Sacha De Carlo, Assistant Professor
Chemistry Department, Marshak Science Building &
New York Structural Biology Center
The City College of New York
Convent Ave & 138th Street
New York, NY 10031
(212) 650-6070
e-mail: sdecarlo at ccny.cuny.edu
URL: http://www.planetesacha.com
================================================

--- On Thu, 2/5/09, Eduardo Sanz Garcia <esanzgar at gmail.com> wrote:

From: Eduardo Sanz Garcia <esanzgar at gmail.com>
Subject: [3dem] Alkylamine vapor while glow discharge
To: 3dem at ncmir.ucsd.edu
Date: Thursday, February 5, 2009, 9:46 PM

The use of alkylamine vapors in glow discharge apparatus makes the
grid positively charged. But I found some contradictory information
about the hydrophobicity:
Some say that it makes the surface hydrophilic, others hydrophobic.
If it makes the surface hydrophobic, while is still used in cryoEM?
Shouldn't that prevent the fluid from spreading evenly on the grid
surface?
Does someone have experience in this type of treatment?

Additionally,
Has someone use magnesium acetate, lysine or polylysine after glow
discharge to make the surface positively charged?

Thank you.
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