Preparation of ATP13A2 cryo-EM grids
Sue Sim, eunyong_park
Published: 2022-07-20 DOI: 10.17504/protocols.io.q26g74eb9gwz/v1
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Abstract
Preparing cryo-EM grids using purified ATP13A2 samples
Steps
Preparing samples
1.
Spin purified and concentrated ATP13A2 sample (5-7 mg/mL) after SEC at 17000x g,4°C
2.
Keep protein on ice
3.
Plasma clean grids
3.1.
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)
4.
Apply 3 μL of protein sample to grids and plunge freeze
4.1.
Used Vitrobot Mark IV (FEI) operated at 4°C and 100% humidity and Whatman No.1 filter paper to blot samples
4.2.
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.
5.
Store grids in liquid nitrogen until ready for imaging.
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