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 supplemented supernatant on ice for one hour, mixing by inversion every 15 minutes. Transfer the tube to a 37 degrees Celsius water bath, and incubate for 10 minutes to induce phase separation.

Centrifuge the sample at 10,000 times g for 10 minutes at room temperature to maintain biphasic separation. Gently pipette off the upper aqueous phase and discard. Add ice-cold TBSE to the lower surfactant phase to refill the tube to its original volume and invert to mix.

Incubate on ice for 10 minutes. Following incubation, transfer the tube to a 37 degrees Celsius water bath and incubate for 10 minutes to induce phase separation, then centrifuge at 10,000 times g for 10 minutes at room temperature. After repeating these steps once more for a total of three separations, remove the upper aqueous phase and discard.

Remove the pellet of precipitated proteins that formed during the course of the extractions by adding one volume of ice-cold TBSE to the surfactant phase. Centrifuge at four degrees Celsius and 16,000 times g for two minutes to pellet the insoluble protein. Immediately transfer the supernatant to a fresh 2.0-milliliter microcentrifuge tube containing 1,250 microliters of 100% acetone.

Mix the sample by inversion and incubate overnight at minus 20 degrees Celsius to precipitate the protein. The next day, centrifuge the sample at 16,000 times g for 20 minutes at room temperature to pellet the lipoproteins with attention to the orientation of the tube. Wash the pellet twice with 100% acetone before decanting the acetone and allowing the sample to air dry.

Then, add 20 to 40 microliters of 10-millimolar Tris-HCL, pH 8.0, and thoroughly resuspend the pellet by pipetting up and down against the wall with the precipitated lipoproteins.