Enrichment of Bacterial Lipoproteins Using Non-Ionic Detergent Phase Separation

Take a bacterial lysate containing hydrophilic proteins, lipoproteins, and protein aggregates.

Add a cold, non-ionic detergent capable of phase separation at warmer temperatures.

Incubate on ice and mix periodically. The detergent binds to the lipoprotein's lipid moiety, forming protein-detergent complexes.

Warm the mixture to induce phase separation. Lipoproteins concentrate in the detergent phase, while hydrophilic proteins remain in the aqueous phase.

Centrifuge at room temperature to maintain the biphasic separation.

Discard the aqueous phase, add a cold buffer to the detergent phase, and mix.

Repeat the cooling, warming, and centrifugation cycle to enrich lipoproteins.

Then, add cold buffer and centrifuge at low temperature to pellet the non-lipoprotein aggregates.

Transfer the supernatant to a tube containing a protein-precipitating solvent and incubate at sub-zero temperature to precipitate lipoproteins.

Centrifuge at room temperature and discard the supernatant.

The precipitated lipoproteins are ready for downstream studies.

Supplement the supernatant with TX-114 surfactant to a final concentration of 2% by adding an equal volume of 4% surfactant in ice-cold TBSE. Incubate the supplem