[3dem] 3dem Digest, Vol 121, Issue 43

[Alexey Amunts]

Hi Sabrina,

Another option is calcium deposits, these are rather idiopathic and localised to mitochondria, but were also reported to be enriched in the cytoplasm of cells from specific compartments such as ovarian follicles for example. To get rid of these random dots, selective precipitation with PEG worked for us, particularly addition of 1,5% PEG 6000 in pH 7,5 buffer containing 100mM KCL, 10min incubation. Not as a last step though, as PEG is a cryoprotectant.

Best wishes,
Alexey

> On 30 Sep 2017, at 05:53, 3dem-request at ncmir.ucsd.edu wrote:
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>   1. Electron dense contamination on cryo grids (Sabrina Pospich)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Fri, 29 Sep 2017 20:52:22 +0000
> From: Sabrina Pospich <Sabrina.Pospich at mpi-dortmund.mpg.de>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: [3dem] Electron dense contamination on cryo grids
> Message-ID: <56377843-1833-48B2-A6E2-19B865A58A03 at mpi-dortmund.mpg.de>
> Content-Type: text/plain; charset="us-ascii"
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> Dear EM community,
> 
> we are currently struggling with an electron dense contamination on our cryo-EM grids (see images below - left panel volta phase plate images with different phase shift - right panel standard TEM images at different magnifications).
> The dots seem to appear almost randomly and like to stick to protein. We furthermore see them independently of the glow discharger (GloCube or an older device) and plunger (Cp3 or Vitrobot) we use and also for both C-flat and Quantifoils grids.
> Has anyone seen those black dots before? We appreciate any hint.
> 
> Cheers,
> Sabrina Pospich
> (Representative for the Raunser lab, MPI Dortmund)
> 
> [cid:115172A7-0078-461B-88F7-50ABF840BA2F]
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