Authors
Creative Biogene
Summary
The outer membrane of gram-negative bacteria contains lipopolysaccharide (LPS), which is a low molecular weight carbohydrate with a molecular mass of 10-20 kDa. LPS is a tripartite molecule consisting of lipid A that is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell. Lipid A has multiple fatty acids which serve to anchor LPS into the bacterial membrane, allowing the O antigen and core oligosaccharide to protrude, and contributes to the main part of the toxicity of gram-negative bacteria. Also known as endotoxin, when consumed by animals, LPS induces a strong inflammatory response and/or sepsis.
Here, we describe a hot aqueous-phenol method for the isolation and purification of LPS from gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acid
Introduction
The Workflow of LPS Extraction
• Reagent Preparation
Prepare 2x SDS buffer. Make the 50 mL solution of 4% β-mercaptoethanol (BME), 4% SDS and 20% glycerol in 0.1 M Tris-HCl, pH 6.8. Add a little of bromophenol blue to dye the solution. Make a 1x SDS-buffer stock by diluting 2X stock 1:1 in sterile distilled H2O. This can be stored at room temperature.
Make three separate 10 mg/mL solutions of DNase I, RNase, and Proteinase K in sterile distilled H2O.
• Preparation of Bacteria for LPS Extraction
Grow an isolated culture of bacteria overnight on Luria Broth (LB) media plate at 37°C. Next day, pre-weigh a 15 ml conical tube, then add 12 ml cold PBS, pH 7.2. Sweep the bacteria growth from an LB media plate with a sterile swab and resuspend in cold PBS.
Determine the concentration of bacteria in solution by evaluating the turbidity of culture with a Spectrophotometer: remove 1 ml of bacteria suspended in PBS and add to the cuvette. Place cuvette in Spectrophotometer and measure OD600 nm. Ensure that the OD600 nm ≥0.6.
Centrifuge conical tube at 2500 x g for 10 minutes to pellet the bacteria. Decant supernatant and repeat centrifugation. Remove supernatant with the pipette and discard. The pellet can be stored at -20 °C, if LPS is not be extracted immediately.
Procedure
Extraction of LPS
Resuspend the pelleted bacteria in 200 μl of 1x SDS-buffer. Ensure that the pellet is completely resuspended through pipetting the solution up and down and slowly. Do not vortex.
Boil the suspended bacteria in a water bath for 15 minutes. Allow the solution to cool at room temperature for 15 minutes.
Add 5 μl of both DNase I and RNase solutions. Incubate the samples at 37 °C for 30 minutes. Next, add 10 μl of the Proteinase K solution. Incubate the samples at 59 °C for 3 hours.
Add 200 μl of ice-cold Tris-saturated phenol to each sample (if Tris-saturated phenol is not available, water saturated phenol may be used as a substitute). Ensure that the caps on the tubes are closed tightly, and vortex each sample for about 5 to 10 seconds.
Incubate the samples at 65 °C for 15 minutes, vortexing occasionally. After incubating cool to room temperature, then add 1 mL of room-temperature diethyl ether to each sample and vortex for 5 to 10 seconds. (Note: Be sure to perform handle ether in a fume hood, because it is volatile.)
Centrifuge the samples at 20,600x g for 10 minutes. Carefully remove the samples from the centrifuge and extract the bottom blue layer. Be sure to avoid the upper, clear layer.
Re-extract the samples by repeating steps 4-6. Two extractions are usually sufficient. If the samples appear cloudy, more extractions can be performed. Add 200 μl of 2x SDS-buffer to each of the extracted samples before separating by SDS-PAGE. 5 to 15 μl of LPS prepared using this method is usually sufficient for visualization.
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