Membrane Proteins | Micelle-free.

GraDeR is an easy protocol for preparing membrane proteins in a micelle free solution.

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How it works

Three easy steps

1. Detergent Exchange

grader_step1

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

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

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.

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