Enrichment of Bacterial Lipoproteins Using Non-Ionic Detergent Phase Separation

Abstract

Source: Armbruster, K. M., Meredith, T. C. Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry. J. Vis. Exp. (2018)

This video demonstrates the enrichment of bacterial lipoproteins using a non-ionic detergent that undergoes phase separation upon warming. Through sequential temperature shifts and centrifugation steps, lipoproteins are selectively partitioned into the detergent phase, while non-lipoprotein contaminants are removed. The enriched lipoproteins are then precipitated, washed, and resuspended for downstream applications.

Protocol

1. Enrichment of Lipoproteins by Triton X-114 (TX-114) Phase Partitioning

1. Supplement the bacterial lysate with TX-114 surfactant to a final concentration of 2% (vol/vol) by adding an equal volume of 4% (vol/vol) surfactant in ice-cold Tris-buffered saline/ Ethylenediaminetetraacetic acid (TBSE) and incubate on ice for 1 h, mixing by inversion every ~15 min.
   NOTE: When chilled, the supernatant and surfactant will be miscible.
2. Transfer the tube to a 37 °C water bath and incubate for 10 min to induce phase separation. Centrifuge the sample at 10,000 x g for 10 min at room temperature to maintain bi-phasic separation.
3. 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 it to mix. Incubate on ice for 10 min.
4. Transfer the tube to a 37 °C water bath and incubate for 10 min to induce phase separation, then centrifuge at 10,000 x g for 10 min at room temperature.
5. Repeat steps 1.3-1.4 once more for a total of 3 separations. Remove the upper aqueous phase and discard.
6. Remove pellet of precipitated proteins that formed during the course of extractions (visible at the bottom of the tube), by adding 1 volume of ice-cold TBSE to the surfactant phase. Centrifuge at 4 °C at 16,000 x g for 2 min to pellet insoluble protein.
   NOTE: Sample should remain a single phase. If phase separation occurs, re-chill sample and centrifuge again. The pellet is generally composed of non-lipoprotein contaminants but can be retained for further analysis.
7. Immediately transfer the supernatant to a fresh 2.0-mL microcentrifuge tube containing 1250 μL of 100% acetone. Pipette up and down thoroughly to wash the sample from the tip as it will be viscous. Mix by inversion and incubate overnight at -20 °C to precipitate protein.
8. Centrifuge the sample at 16,000 x g for 20 min at room temperature to pellet lipoproteins with attention to the orientation of the tube. Lipoproteins will form a thin, white film along the outside wall of the tube.
9. Wash the pellet twice with 100% acetone. Decant acetone and allow sample to air dry.
10. Add 20-40 μL of 10 mM Tris-hydrochloride, pH 8.0 or a standard Laemmli SDS-PAGE sample buffer and thoroughly resuspend by pipetting up and down against the wall with the precipitated lipoproteins. Scrape the side of the tube with the pipette tip to dislodge lipoproteins.
    NOTE: Lipoproteins will not dissolve in Tris buffer but rather form a suspension.
    1. Store lipoproteins at -20 °C until use.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetone	EMD	AX0116-6
Ethylenediaminetetraacetic acid (EDTA)	Fisher Scientific	BP118
Tris-hydrochloride (HCl)	Fisher Scientific	BP152
Triton X-114	Sigma-Aldrich	93422
Sodium chloride	Macron Fine Chemicals	7581-06