[3dem] Re: 3dem Digest, Vol 42, Issue 8

[DocDave50 at aol.com]

Reza,
 
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 
surface.
 
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:

Hi,
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.

Sincerely,

Reza


-------------- next part --------------
An HTML attachment was scrubbed...
URL: https://mail.ncmir.ucsd.edu/mailman/private/3dem/attachments/20110215/8119d15b/attachment.html