PEPTERGENT Extraction Method - Direct extraction of membrane proteins without conventional detergents

Protocol overview: Incubate membranes with Peptergent (e.g., 1:1 Peptergent:protein, w/w), centrifuge at 100,000 × g to remove insoluble material, and collect the supernatant containing Peptergent-solubilized membrane proteins.

PEPTIDISC “In-Gel” Method - Determine the optimal peptide:protein ratio for membrane protein solubilization

Protocol overview: Prepare detergent-solubilized membrane protein with a range of peptide:protein ratios (e.g., 0–4:1, w/w). Analyze by CN-PAGE to assess solubilization and complex integrity, then select the optimal ratio for subsequent Peptidisc preparation.

PEPTIDISC “On-Column” Method - Rapid preparation of purified Peptidisc complexes

Protocol overview: Combine Peptidisc peptide and the target membrane protein at the optimal ratio determined using the “In-Gel” method. Separate the mixture by size-exclusion chromatography (SEC), then collect the Peptidisc fraction, free from detergent, protein aggregates, and excess peptide. 

PEPTIDISC “On-Gradient” Method - Large-scale preparation of purified Peptidisc complexes

Protocol overview: Combine Peptidisc peptide and the target membrane protein at the optimal ratio determined using the “In-Gel” method. Load the mixture onto a linear sucrose gradient (e.g., 4–12%) and ultracentrifuge overnight in a swinging-bucket rotor. Collect the Peptidisc fractions, free from protein aggregates, detergent, and excess peptide.

PEPTIDISC “On-Beads” Method - Trap membrane proteins in Peptidisc during affinity purification

Protocol overview: Bind the target membrane protein to affinity beads (e.g., Ni-NTA) and wash away protein contaminants and excess detergent. Add Peptidisc peptide in the presence of a low (CMC) concentration of detergent, then wash away the remaining detergent and recover excess peptide for reuse. Finally, elute the Peptidisc-trapped membrane protein in detergent-free buffer.