Separation of Inner and Outer Membrane Fractions of Gram-Negative Bacteria

Abstract

Source: Cian, M. B. et. al., Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii. J. Vis. Exp. (2020)

This video demonstrates the separation of the inner and outer membrane fractions of Gram-negative bacteria using sucrose gradient ultracentrifugation. The membrane fractions are purified and stored at low temperatures for targeted analysis of membrane-specific components.

Protocol

1. General Reagents and Media Preparation for Membrane Extraction

1. Bacterial growth media: Prepare and sterilize 1 L of broth media in a thoroughly cleaned and autoclaved 2 L flask.
2. General resuspension buffer (1 M tris(hydroxymethyl)aminomethane buffer (Tris Buffer) pH 7.5; 50 mL): Dissolve 6.05 g of Tris base in 30 mL of H₂O. Adjust pH to 7.5 with 5 M HCl. Adjust the final volume to 50 mL with ultrapure H₂O.
3. Master stock of divalent cation chelation solution (0.5 M ethylenediamine tetraacetic acid, EDTA pH 8; 100 mL): Add 18.6 g of disodium ethylene tetraacetate·2H₂O to 80 mL of H₂O. Stir vigorou....

Results

Figure 1: A schematic depicting the Gram-negative bacterial membrane isolation procedure described in this meth.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 L Centrifuge bottles, PC/PPCO, super speed, with sealing cap, Nalgene	VWR	525-0466
1 L Pyrex media storage bottle with high temperature cap	VWR	10416-312
2000 mL Erlenmeyer flask, narrow mouth	VWR	10545-844
4-20% mini PROTEAN precast protein gels, 12 well	BIORAD	4561095
4x Laemmli sample buffer 10 mL	BIORAD	1610747
50 mL sterile polypropylene centrifuge tubes	VWR	89049-174
7 mL Dounce tissue homogenizer with two glass pestles	VWR	71000-518
70 mL polycarbonate bottle assembly	Beckman Coulter Life Sciences	355622
Agar powder	VWR	A10752
B10P benchtop pH meter with pH probe	VWR	89231-664
Barnstead GenPure xCAD Plus UV/UF - TOC (bench version)	ThermoFisher Scientific	50136146
Benzonase nuclease	MilliporeSigma	70746-3
EDTA disodium salt dihydrate 99.0-101.0%, crystals, ultrapure bioreagent molecular biology grade, J.T. Baker	VWR	4040-01
EmulsiFlex-C3	Avestin, Inc.
Fiberlite F13-14 x 50cy fixed angle rotor	ThermoFisher Scientific	75006526
Hydrochloric acid 6.0 N	VWR	BDH7204
IBI scientific orbital platform shaker	Fischer Scientific	15-453-211
LB Broth Miller	VWR	214906
Lysozyme, Egg white, ultra pure grade	VWR	VWRV0063
Magnesium chloride hexahydrate	Sigma Aldrich	M2670
Optima XPN-80 - IVD	Beckman Coulter Life Sciences	A99839
Protease -50, EDTA free	G Biosciences	786-334
Sodium hydroxide ≥99.99%	VWR	AA45780-22
Sorval RC 6 Plus centrifuge	ThermoFisher Scientific	36-101-0816
Sucrose	MilliporeSigma	SX1075-3
SW 41 Ti swinging-bucket rotor	Beckman Coulter Life Sciences	331362
Tris(hydroxymethyl)aminomethane (TRIS, Trometamol) ≥99.9% (dried basis), ultrapure Bioreagent Molecular biology grade, J.T. Baker	VWR	JT4109-6
Type 45 Ti fixed-angle titanium rotor	Beckman Coulter Life Sciences	339160
Ultra-clear tube ,14 x 89mm	Beckman Coulter Life Sciences	344059
Vortex-Genie 2	VWR	102091-234