[3dem] Lack of Lateral Views

[Reinhard Rachel]

>>> 05.10.2012 um 19:15:
> I am working on a multimeric membrane protein that has a strong  
> tendency to form rings. The top views of these rings can be visualized  
> rather easily using negative-staining EM. However, I am unable to find  
> ANY lateral views. Could someone suggest a solution? CryoEM is not  
> feasible for me at the moment.
> I use Uranyl Acetate for staining over '300 mesh' carbon (pre)coated  
> copper grids.

dear Veer,
different treatments of the carbon film ("hydrophobic", i.e. ageing and no hydrophilization; vs. hydrophilization = glow discharge with 'no' additive; glow discharge with pentylamine; Aebi+Pollard 1987; Dubochet ...). Other stains. Combination of these "tricks". Changing the pH of the solution of the protein (! 4,5,6,7,8,9 ...) may alter the aggregation state and/or the adsorption. 
In fact, you also get some kind of 3D info from negatively stained samples by collecting 3D information using the random conical tilting (check M.Radermacher's papers), and/or by tomography. Yes, it is limited due to staining and air-drying, but it is more than the projection, only. 
kind regards,
Reinhard

-- 
Prof. Dr. Reinhard Rachel
University of Regensburg
Centre for EM / Anatomy
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