May 03, 2024
Transmission Electron Microscopy of Native Nanodiscs
- Caroline Brown1,2,3,
- Snehasish Ghosh1,2,3,
- Kallol Gupta1,2,3
- 1Nanobiology Institute, Yale University, West Haven, CT, USA;
- 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA;
- 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Caroline Brown, Snehasish Ghosh, Kallol Gupta 2024. Transmission Electron Microscopy of Native Nanodiscs. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92ydml3e/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: May 03, 2024
Last Modified: May 03, 2024
Protocol Integer ID: 99194
Abstract
This is a protocol for conducting transmission electron microscopy of native nanodiscs to determine population size distribution and morphology.
Staining grids
Staining grids
32m 35s
Dilute nanodisc samples into single molecule range, typically around between 1:10 and 1:100 is the samples have been through size exclusion chromatography using dilution buffer (50 millimolar (mM) Tris HCl pH 7.4) .
Glow-discharge carbon-coated copper grids (200 mesh) for 00:00:30 seconds.
30s
Apply 5 µL sample to the grid and after 00:01:00 minute blot off with Whiteman ashless filter paper.
1m
Wash grid once with 5 µL uranyl formate for 00:00:05 seconds.
5s
Stain grid with 5 µL uranyl formate for 00:01:00 minute and blot dry with Whiteman ashless filter paper.
1m
Allow to dry for 00:30:00 minutes to Overnight
30m
Image Acquisition
Image Acquisition
Take micrographs using a JEOL JEM 1400PLUS electron microscope at an operating voltage of 80 kV.