PEPTERGENT Extraction Method

1. Incubate membranes with an excess of Peptergent (e.g. 1:1 (w/w) ratio).

2. Centrifuge 100,000 x g.

3. Collect supernatant.

PEPTIDISC Reconstitution Methods

The "in-gel" method - to rapidly determine the optimal peptide to protein ratio

1.    Combine the purified membrane protein with increasing amounts of peptides.

2.    Load on a native gel, run electrophoresis, and stain the gel.

3.    Determine what the optimal peptide to protein ratio is (i.e., last right lane on the gel below).

The "on-column" method - to rapidly get pure peptidisc preparations

1.    Combine peptide and target membrane protein at a ratio determined via the "on-gel" method.

2.    Load mixture on size exclusion chromatography.

3.    Collect peptidiscs, free from detergent, protein aggregates and excess peptides.

The "on-gradient" method - for large-scale peptidisc preparations

1.    Combine peptide with target membrane protein at a ratio determined via the "on-gel" method.

2.    Load on linear sucrose gradient (e.g 4%-12%).

3.    Ultra-centrifuge overnight in a swinging rotor.

4.    Collect peptidiscs, free from protein aggregates, detergent and excess peptides.

The "on-beads" method - to trap membrane protein in peptidisc during purification 

1.    Bind the target membrane protein on affinity beads (e.g., Ni-NTA).

2.    Wash away protein contaminants and excess detergent.

3.    Add peptide with a low (CMC) level of detergent. 

4.    Wash away detergent and recover excess peptide (it can be reused).

5.    Elute trapped membrane protein in detergent-free buffer.