Free detergent impairs biophysical methods. GraDeR removes detergent so you can treat your membrane proteins like cytosolic samples.
Easy to use
The protocol is simple and robust. All you need is the Lauryl Maltose Neopentyl Glycol (LMNG) detergent and an ultracentrifuge.
GraDer works well and reliable with almost any protein and detergent in a large variety of buffers and conditions.
How it works
Three easy steps
1. Detergent Exchange
Simply add LMNG to your protein in your extraction detergent solution. Because of the low off-kinetics of LMNG, the detergent exchange at the bicelle will automatically occur. Has been tested successfully with most extraction detergents and works within 2 hrs at 4°C-room temperature.
2. Gradient centrifugation
After detergent exchange, proteins are subjected to a gradient centrifugation in a reciprocal gradient of decreasing LMNG concetrations (max. 0.01%-0%) and increasing crowding agent concentration (we use glycerol). Gradient fractions which are harvested at the bottom third will be free of detergent micelles.
3. Crowding Agent Removal
The crowding agent can be removed by size exclusion chromatography (i.e. desalting columns), dialysis for sensitive samples and other methods to obtain a sample in the buffer of choice. Concentrators work well after grader, too: samples show increased stability while maintaining functional flexibility.