Jul 20, 2022
Preparation of ATP13A2 cryo-EM grids
- Sue Sim1,
- eunyong_park 1
- 1University of California, Berkeley
Protocol Citation: Sue Sim, eunyong_park 2022. Preparation of ATP13A2 cryo-EM grids. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74eb9gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 17, 2022
Last Modified: July 20, 2022
Protocol Integer ID: 66879
Keywords: atp13a2 cryo, atp13a2 sample, preparing cryo, cryo
Abstract
Preparing cryo-EM grids using purified ATP13A2 samples
Troubleshooting
Preparing samples
10m
Spin purified and concentrated ATP13A2 sample (5-7 mg/mL) after SEC at 17000 x g, 4°C, 00:10:00
10m
Keep protein on ice
Plasma clean grids
Used gold holey carbon grids (Quantifoil R 1.2/1.3, 400 mesh) and PELCO easiGlow Glow Discharge Cleaning System (0.39 mBar, 25-30 mA, 40-45 sec)
Apply 3 μL of protein sample to grids and plunge freeze
Used Vitrobot Mark IV (FEI) operated at 4°C and 100% humidity and Whatman No.1 filter paper to blot samples
Our blotting settings were 3.5-4.5 second blot at force 25, but every Vitrobot is slightly different. Use optimal settings for your machine.
Store grids in liquid nitrogen until ready for imaging.