Dear Erik
We have used the "rapid flush" method for negative stain EM.
This method requires higher concentration of proteins than the
conventional negative stain EM method.
Please refer to Imai et al., 2015 Nature Communications 6:8179. In
Methods, I hope that you would find the "rapid flush" method in the
section of "Negative stain EM of dynein in the absence of MTs".
I hope that this information helps your research.
Best wishes,
Hiroshi
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Hiroshi Imai (PhD)
Laboratory of Cell Motility,
Department of Biological Sciences, Graduate School of Science,
Osaka University,
Machikaneyama-cho 1-1, Toyonaka, Osaka, 560-0043, Japan
E-mail: <[log in to unmask]>
_______________________________
On 2018/07/13 6:48, Erik Klontz wrote:
> Hi CCPEM community,
>
> I'm trying to solve the structure of a 140kDa complex (90+50) that has a relatively weak binding affinity (~2uM). I was going to start with negative stain, but I've found that at the low concentrations needed for negative stain, my complex falls apart. I've tried to crosslink it using GraFix and other methods, but have had very limited success crosslinking the complex without additional artifacts. This leaves me wanting to do negative stain at high protein concentrations (~2+ mg/ml). Does anyone know tricks for adsorbing FEWER particles to a grid so I can get away with using higher concentrations?
>
> Thanks!
> Erik
>
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