Database-driven protein solubilization
Caroline Brown, Snehasish Ghosh, Kallol Gupta
Abstract
This is a protocol for using the extraction database found at www.polymerscreen.yale.edu for optimal extraction of membrane proteins into native nanodiscs.
Steps
Choose your protein of interest.
Visit www.polymerscreen.yale.edu to search for your protein in the extraction database. This will provide you with the polymer to use for optimal protein extraction.
As an example, below are representative organellar markers and the optimal polymer for extraction:
TMEM192 --> AASTY650
TGN46 --> ChloroSMA60
OMP25 --> SMA200
VAPA --> AASTY650
KRas --> ChloroSMA80
Once the optimal polymer has been determined, express the protein of interest, or harvest cells for endogenous solubilization.
Resuspend cellular membranes in the chosen polymer and incubate at 4°C with rotation for 2h 0m 0s hours.
Ultracentrifuge samples ay 200,000xg for 1h 0m 0s hours to remove insoluble material.
