May 03, 2024
Step-centrifugation Assay
- 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. Step-centrifugation Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqn75xgk5/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: 99142
Keywords: centrifugation assay this protocol detail, centrifugation assay, vesicles from native nanodisc, vesicle, assay, speed for full separation, native nanodisc
Abstract
This protocol details the protocol for conducting a step-centrifugation assay to determine the minimum required speed for full separation of vesicles from native nanodiscs.
Troubleshooting
Resuspend cells in lysis buffer (50 millimolar (mM) Tris HCl pH 7.4 , 150 millimolar (mM) NaCl , 10 % volume glycerol ) and lyse using nitrogen cavitation (750 PSI for 00:15:00 minutes).
15m
Centrifuge lysed cells at 4000rpm for 00:10:00 minutes to pellet cell debris.
10m
Spin the supernatant in a series of sequential ultracentrifugation steps at speeds of 0 µL 20,000xg, 100,000xg, 150,000xg, and 200,000xg with 01:00:00 hour for each spin.
1h
After each spin subject the sample to dynamic light scattering to calculate population size distribution.