[3dem] re: uneven staining/contamination on negative stained grids

[Vince Ramey]

Berith,

I've run into all those phenotypes too!  Here's my take:

Image #1 looks like the stain is just too thin.  Basically the carbon is
totally dry.  The sample is absorbing stain and that's why it looks
uneven.  This happens to me easily and can happen on grids where the other
side of the grid looks great.  I'd say this is also happening in some
degree to Image #4.

The middle two however look a lot like a problem I had and seemed to solve
by thinking a lot about hydrophobicity.  That blotchiness went away
entirely when I:

1) made the carbon much thicker (better able to charge?)
2) Glow discharged exclusively in an Edwards evaporator.  1 min.
3) Used UFormate made fresh.  This may not be essential.  UAcetate works
ok too, but is less consistent in my hands.  Bad stain usually looks like
lots of dark spots where it's ppt-ing, which isn't happening here.

The biggest thing for me was making the carbon THICK.  Very easily visible
on the mica.  Even with carbon that looked like medium pencil sketching I
got great contrast on a 400 kDa complex so don't worry too much about
thinness for contrast.

Also, I can't say enough about how much more I like C-flats Vs
Quantifoils, although I wouldn't say that's the issue here.  For cryo
there's no comparison.

Good luck!

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Vincent Ramey

Nogales Lab
http://cryoem.berkeley.edu
Phone : (510) 666-3335

Biophysics Graduate Group
http://biophysics.berkeley.edu

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