LPS Extraction Protocol
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 acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction ways.
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.
• 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.
- Davis Jr M R, Goldberg J B. Purification and visualization of lipopolysaccharide from Gram-negative bacteria by hot aqueous-phenol extraction. Journal of visualized experiments: JoVE, 2012 (63).
- Chowdhury F A, et al. Purification, extraction and visualization of lipopolysaccharide of Escherichia coli from urine samples of patients with urinary tract infection. Avicenna Journal of Clinical Microbiology and Infection, 2015, 2(4).