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

Start with a Gram-negative bacterium's total membrane fraction, including inner and outer membrane vesicles, suspended in a low-density sucrose that preserves structural integrity.

To separate the membrane vesicles, sequentially add high-density and medium-density sucrose solutions into an ultracentrifuge tube.

Next, add the membrane fraction and the low-density sucrose to create a discontinuous sucrose gradient.

Ultracentrifuge to separate vesicles based on their densities.

The less-dense inner membrane vesicles form a layer at the top.

The denser outer membrane vesicles, rich in lipopolysaccharides and proteins, form a band at the interface of medium and high-density sucrose solutions.

Collect the inner membrane layer and discard the gradient solution, leaving a small amount.

Then, collect the outer membrane layer.

Add a storage buffer to each fraction for stabilization.

Centrifuge to separate the vesicles and remove the supernatant.

Resuspend the purified membrane vesicles, homogenize, and store them at low temperatures for further analysis.

To prepare the sucrose gradient, hold a polypropylene or Ultra-clear Open-Top tube in a slightly tilted position.

Slowly add two milliliters of the hig