Cryo-EM sample preparation for RCKW:DARPin complex
Marta Sanz Murillo
Abstract
This is Leschziner's Lab protocol for making cryo-EM grids for RCKW:DARPin complex.
Before start
Decide which protein concentration to use, and create the proper LRRK2 buffers in order to obtain the right salt concentration (150 mM NaCl).
Steps
Freezing Grids
Plasma clean grids.
We used UltrAuFoil Holey Gold 1.2/1.3 300 mesh grids and plasma cleaned them in the Solarus II (Gatan) using the QuantiFoil Au preset.
Dilute samples to desired concentration in the LRRK2 buffer . Make sure final salt is at 150 mM NaCl.
For best results, make 8µL samples, good for freezing 2 grids. This is to minimize time spent outside of storage buffer, reducing aggregation.
Apply protein to grids and plunge freeze (3-4 uL)
We used a Vitrobot (FEI) to blot away excess sample and plunge freeze
Store grids in liquid nitrogen until ready for imaging.
