[3dem] Negative stain on graphene oxyde grids

[Kenner, Lillian]

Hi Julian and 3DEM,


I have done negative stain with graphene oxide coated grids and I found it to be very useful. You only have to do a few things differently.

First, I used the protocol in this paper to make my grids: https://www.sciencedirect.com/science/article/pii/S1047847718301655?via%3Dihub  --- I glow discharge these for 10 seconds which is necessary for binding my sample.


Second, I increased the amount of time I use for the final stain drop while making the negative stains grids from 1 min to 2-3 minutes.


And finally, as the grids tear under the beam it helps to lower the spot size to decrease the beam intensity (non-glow discharged grids don't tear as easily).

As you can see from the images below, the negative stain contrast is good over the graphene oxide film even though the sample is ugly. This was very useful to me early in my project when I didn't have all the biochemistry figured out, but I wanted to have some idea of what the concentration would be like on GO grids. I was able to see that small contaminating proteins were binding the graphene oxide surface instead of my large particle. In fact, the contaminating particles are pretty small (30kDa) and I couldn't see them on the thicker carbon coated negative stain grids.


The second picture is the 20S proteasome, which looks great negative stained on GO.


Hope that helps,

Lillian Kenner

[https://lh4.googleusercontent.com/Z0269xiFg4aR2dBQ1-4G9yxMX-Zq0-v-K2koRcXoOJV7O2KoST8yQUi68CJa-LHmWZ5beuOHmQ0SiKXVl8tG13bED6snp7i8YAsiStpmwQY2X0uj558spZ6RrhnAhpo_QZjQAk0XxFA] [https://lh6.googleusercontent.com/zps74QwC0MV6Bwt30xAXJgWgOe9bPeUrrsx5w_zyJOIJ4Royh4B_03eFtwRQAveGycphrwk68T3O9ia0o4sYGT4bLUB1C2OhS0E4WdyM7rBIizQx5ScqMqQAItfxxUquzG3rjQP9BnQ]


A simple and robust procedure for preparing graphene-oxide ...<https://www.sciencedirect.com/science/article/pii/S1047847718301655?via%3Dihub>
www.sciencedirect.com
Graphene oxide (GO) sheets have been used successfully as a supporting substrate film in several recent cryogenic electron-microscopy (cryo-EM) studies of challenging biological m


________________________________
From: 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Matthews-Palmer, Teige Rowan Seal <t.matthews-palmer14 at imperial.ac.uk>
Sent: Wednesday, September 12, 2018 12:07:13 AM
To: Julien Bous
Cc: 3dem at ncmir.ucsd.edu
Subject: Re: [3dem] Negative stain on graphene oxyde grids

Dear Julien,
It’s a good idea, to check that your sample will adsorb to GrOx, and perhaps you’d also like to see how it’s oriented.
However, things might look different between a negative stain protocol versus blotting and plunge freezing. It’s probably better to move quickly to freezing, as that’s where optimisation can take time, and you can see how your sample interacts with GrOx under the conditions you’ll use for eventual data collection. In other words, diagnose GrOx at stage 3 of this scheme: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5140023/figure/F1/
If neg stain on GrOx turns out to be challenging, it will be a waste of time for likely no benefit over regular grids.
Negative stain on regular carbon grids can tell you if your particle looks the right shape & size, and what the concentration is like. (A rule of thumb some use to start, is having found a good concentration in neg stain, 10x for cryo in holes, and a smaller increase for cryo on support films.)

All the best
Teige
On 11 Sep 2018, at 16:55, Julien Bous <julien.bous at etu.umontpellier.fr<mailto:julien.bous at etu.umontpellier.fr>> wrote:


Dear Community,

I'm PhD student in cryoEM and I plan to use graphene oxide grids for a complicate sample. Therefore, in order to make a first assessment of the interaction between the graphene oxide and my sample, I want to try in first to do a negative stain on graphene oxide with my sample Has anyone ever seen or tried this , I didn't find any bibliography? And is it looks like a good idea for more experienced peoples?

Thank you very much for your time.

Best,

Julien

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