[3dem] Electron dense contamination on cryo grids

[Sabrina Pospich]

Dear Alexey, Sameer, Stefan and Ben,

thanks for sharing your ideas with us. We have already suspected the contamination to be gold. However, we hardly use gold fiducial in our lab and even less frequent with the plungers. We will clean our equipment more carefully in the future and hope that solves the problem.

Cheers,
Sabrina

On 30 Sep 2017, at 06:46, Ben Engel <bdengel at gmail.com<mailto:bdengel at gmail.com>> wrote:

I agree with Stefan that they look like Gold fiducials (5nm or 10nm).  In addition to tweezers, if you are using a plunger such as a Vitrobot, it is also possible that the contaminating particles are on the pads where you place the rings of blotting paper.

Best,
Ben

On Sat, Sep 30, 2017 at 12:20 AM, Stefan Bohn <bohn.stefan.acc at gmail.com<mailto:bohn.stefan.acc at gmail.com>> wrote:
Hello Sabrina,

these particles look remarkably similar to nanogold particles I used before. These particles also preferred proteins (they loved the 26S) or showed clusters similar to the ones on the lower right image. They come in sizes from 2 to >5nm (can't see a scale-bar, but it seems your contamination is of similar size).

Is anyone using fiudical markers of any sort in your lab? One source of contamination would be the tweezers that hold the grid during plunging. In case those tweezers are shared among several 'plungers' and not readily cleaned after use of the markers, they could appear on the next grid being plunged.

Hope that helps,
Stefan.

On Fri, Sep 29, 2017 at 1:52 PM, Sabrina Pospich <Sabrina.Pospich at mpi-dortmund.mpg.de<mailto:Sabrina.Pospich at mpi-dortmund.mpg.de>> wrote:
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)

<Screen Shot 2017-09-29 at 16.52.26.png>

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Stefan Bohn
Gladstone Institute
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San Francisco, CA. 94158
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Fax: +1 (415) 514 - 9736<tel:(415)%20514-9736>
stefan.bohn at gladstone.ucsf.edu<mailto:stefan.bohn at gladstone.ucsf.edu>

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