Single Particle Analysis for purified protein complexes.
Workflow:
- Wash holey carbon membrane/ gold grids with pure acetone (as required)
- Natural dry
- Glow discharge the grid by the Quorum GloQube
- Prepare graphene-oxide membrane on the gold grid (as required)
- Graphene oxide dispersion in H2O (Sigma-Aldrich, Merck)
- Quantifoil, C-flat, UltraAuFoil
- Anti-capillary crossover tweezers
- Prepare ultra-thin carbon membrane on the gold grid (as required)
- Carbon coats on mica
- Quantifoil, C-flat, UltraAuFoil
- Put the purified protein-complex on the designed grid as above
- Blot the access volume on the grid with filter papers
- Vitrification purified protein-complexes on the grid by the FEI Vitrobot
- Set the frozen grid on the Gatan cryo-transfer holder
- Insert the cryo-holder to the FEI TalosF200C
- General alignment for low electron-dose imaging
- EPU image acquisition after the Atlas
- Image processing by the Relion or CryoSparc on the Cryo-EM GPU server
- Repeat several refinements
- 3D-model
