Step-centrifugation Assay
Caroline Brown, Snehasish Ghosh, Kallol Gupta
Published: 2024-05-03 DOI: 10.17504/protocols.io.36wgqn75xgk5/v1
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.
Steps
1.
Resuspend cells in lysis buffer (50millimolar (mM), 150millimolar (mM) , 10% volume ) and lyse using nitrogen cavitation (750 PSI for 0h 15m 0s minutes).
2.
Centrifuge lysed cells at 4000rpm for 0h 10m 0sminutes to pellet cell debris.
3.
Spin the supernatant in a series of sequential ultracentrifugation steps at speeds of µL 20,000xg, 100,000xg, 150,000xg, and 200,000xg with 1h 0m 0s hour for each spin.
4.
After each spin subject the sample to dynamic light scattering to calculate population size distribution.
