This article details a method for isolating inner and outer membrane vesicles from a Gram-negative bacterium using a sucrose gradient. The process involves ultracentrifugation to separate vesicles based on density, allowing for the collection of distinct membrane fractions for further analysis.
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 higher density sucrose solution. Then add four milliliters of the lower density sucrose solution. When preparing the density gradient, add the sucrose solution slowly to avoid mixing the layers.
The sucrose layers should be slightly visible and appear generally defined. Next, add the total membrane fraction which was previously resuspended in one milliliter of low density, isopycnic sucrose solution. Finally, fill the remainder of the tube by adding approximately six milliliters of the low density, isopycnic sucrose gradient solution.
Ultracentrifuge the samples using a swinging bucket rotor at 288,000 times G and four degrees Celsius for 16 to 23 hours. Cut the end off a P1000 pipette tip about five milliliters from the point. After centrifugation is complete, use the pipette to remove the upper, brown layer from the tube.
Transfer this layer, which contains the inner membrane fraction, to a polycarbonate bottle for ultracentrifugation. Leave about two milliliters of the sucrose solution above the interface between the 53% sucrose and the 73% sucrose to ensure that the lower, white, outer membrane fraction does not cross contaminate the inner membrane fraction. Again using a P1000 pipette tip with the end removed, remove the outer membrane layer and transfer it to a polycarbonate bottle.
Fill the remaining void of polycarbonate bottle with isolated membrane storage buffer and mix by inversion or pippetting. Retain the samples on ice. To collect the membranes, ultracentrifuge the polycarbonate bottles at 184,500 times G and four degrees Celsius for one hour.
Finally, discard the supernatants and add 500 to 1,000 microliters of storage buffer. Resuspend the membranes by Dounce homogenization. Collect the samples in two milliliter microcentrifuge tubes.
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