[3dem] Re: 3dem Digest, Vol 42, Issue 8
DocDave50 at aol.com
DocDave50 at aol.com
Tue Feb 15 20:18:08 PST 2011
One of the issues with glycosylated proteins has to do with the hydration
of the glycolytic tree. These proteins should stain well but possibly not
with Uranyl Acetate at pH 5.5.
I have used 3-4% AmMolybdate titrated to pH 7.4 and included 0.5-1%
Trehalose, pH 7.4. The way that the solutions were prepared is as follows:
6% AmMolyb prepared at pH 7.4
2% Trehalose prepared in your favorite buffer titrated to pH 7.4
Just prior to use, a 1:1 mixture of both is prepared (rationale: the
Trehalose tends to oxidize the Molybdate such that the solution turns
blue...no good for staining)
Using reasonably thick continuous carbon (There was a recent 3DEM note
which suggested that thicker carbon yields better Negative Stain results.
Their suggestion was that there is less charging affects with the thicker
carbon), you would use the usual 1 minute protein adsorption. When you are
removing this first solution containing your protein, leave a thin layer of
liquid on the grid...don't blot dry (this keeps the protein from degrading
due to dehydration affects). Then add the Molybdate mixture for 1 minute,
wick off the stain and ensure that the liquid is thoroughly removed (at this
point, the Trehalose fulfills the hydrogen bonding needs in both the protein
and carbohydrate tree).
The way I have prepared my thicker carbon is by many (30-40) 2 second
carbon evaporation steps on freshly cleaved mica. The benefit of this approach
is that the carbon surface upon which you are putting your sample is very
smooth and very strong due to the multi-layered approach. I have found
that 45 seconds at 25 mA glow discharge provides a very nicely charged carbon
I hope this helps with your negative staining issue.
David W. Chester, Ph.D.
In a message dated 2/15/2011 3:00:19 P.M. Eastern Standard Time,
3dem-request at ncmir.ucsd.edu writes:
Does anyone know if glycosylated proteins will stain differently
than non-glycosylated proteins? If so, is there a particular stain
that I should be using? Essentially, I don't see any particles and
the protein is large enough for EM. Thanks for the help.
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