Purified TRPC5 protein sample (3.5 μl) in PMAL-C8 at 7.0 mg/ml was applied to glow-discharged Quantifoil R1.2/1.3 holey carbon 400-mesh copper grids (Quantifoil). Grids were blotted for 7 s at 100% humidity and flash frozen by liquid nitrogen–cooled liquid ethane using a FEI Vitrobot Mark I (FEI). The grid was then loaded onto FEI TF30 Polara electron microscope operated at 300-kV accelerating voltage. Image stacks were recorded on a Gatan K2 Summit direct detector (Gatan) set in super-resolution counting mode using SerialEM (35), with a defocus range between 1.5 and 3.0 μm. The electron dose was set to 8 e−/physical pixel per s, and the subframe time was set to 0.2 s. A total exposure time of 8 s resulted in 40 subframes per image stack. The total electron dose was 42.3 e−/Å2 (~1.1 e−/Å2 per subframe).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.