May 03, 2024

Public workspaceStep-centrifugation Assay

DOI
dx.doi.org/10.17504/protocols.io.36wgqn75xgk5/v1
  • 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
Caroline Brown
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgqn75xgk5/v1
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 02, 2024
Last Modified: May 03, 2024
Protocol Integer ID: 99142
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.
Resuspend cells in lysis buffer (Concentration50 millimolar (mM) Tris HCl pH 7.4 , Concentration150 millimolar (mM) NaCl , Concentration10 % volume glycerol ) and lyse using nitrogen cavitation (750 PSI for Duration00:15:00 minutes).

15m
Centrifuge lysed cells at 4000rpm for Duration00:10:00 minutes to pellet cell debris.

10m
Spin the supernatant in a series of sequential ultracentrifugation steps at speeds of Amount0 µL 20,000xg, 100,000xg, 150,000xg, and 200,000xg with Duration01:00:00 hour for each spin.

1h
After each spin subject the sample to dynamic light scattering to calculate population size distribution.