The overall goal of this procedure is to extract LPS from Gram-negative bacteria. This is accomplished by first growing a five milliliter overnight culture of bacteria in appropriate medium and adjusting the culture to an optical density at 600 nanometers of 0.5. Next, the bacteria is pelleted by micro centrifugation and the cells are resuspended in a tris buffer containing SDS and beam or capto ethanol.
Then the cell lysates are boiled and treated with protease and nuclease. Finally, two sequential extractions are carried out with hot aqueous phenol and dyl ether carefully remove and retain the aqueous blue layer. Ultimately, results can be obtained that show amount as well as the presence or absence of LPS and strains of bacteria, as well as O antigen chain length modality through separation by SDS page and western blood analyses with LPS specific antibodies or direct staining of the polysaccharides in the gel.
The advantage of this technique compared to the protease treatment of Hitchcock and Brown is that this technique leads to pure LPS samples that can be better resolved by SDS page and western blotting. This method can provide insight into the synthesis and regulation of lipopolysaccharide in VIR into virtually any gram-negative bacteria, including e coli and salmonella, as well as biodefense and emerging pathogens such as Ella and Acetobacter respectively To grow bacteria. For LPS extraction, set up an overnight culture of gram-negative bacteria in five milliliters of LB supplemented with antibiotics.
If necessary, grow the culture overnight in a shaking incubator at 200 RPM and 37 degrees Celsius. Use LB to dilute the culture one to 10, and then measure the optical density at 600 nanometers based on the result. Prepare a 1.5 milliliter suspension of the bacteria with a final optical density at 600 nanometers of 0.5.
Pellet the bacteria in a micro centrifuge at 10, 600 times G for 10 minutes. Then remove and discard the supernatant. To extract LPS prepare the following solutions, one XSDS buffer, 4%betta me capto ethanol or BME 4%SDS, and 20%glycerol in 0.1 molar tris HCL pH 6.8 with a pinch of bromo phenol blue and 10 milligrams per milliliter solutions of DNA one RNAs and proteinase K in sterile distilled water resuspend.
The pelleted bacteria in 200 microliters of one XSDS buffer ensure that the pellet is completely resuspended by pipetting the solution up and down. Slowly boil the suspended bacteria in a water bath for 15 minutes. Then allow the solution to cool at room temperature for 15 minutes.
As an optional step, add five microliters of each of the DNA one and RNA solutions and incubate the samples at 37 degrees Celsius for 30 minutes. Add 10 microliters of the protease K solution and incubate the samples at 59 degrees Celsius for three hours. Next to each sample, add 200 microliters of ice cold tris, saturated phenol.
Close the caps tightly and vortex each tube for five to 10 seconds. Incubate the samples at 65 degrees Celsius for 15 minutes. Vortexing, occasionally cool them to room temperature.
Then under a fume hood, add one milliliter of room temperature, ethyl ether to each sample and vortex for five to 10 seconds. Centrifuge the samples at 20, 600 times G for 10 minutes and carefully remove them from the centrifuge. Extract the bottom blue layer from each sample, avoiding the upper clear layer.
Add an additional 200 microliters of ice cold tris, saturated phenol, and repeat the extraction to each tube. Add 200 microliters of two XSDS buffer. Separate five to 15 microliters of each LPS sample on eight to 15%SDS poly acrylamide gels.
Shown here is a 12%SDS page gel of LPS samples prepared from different strains of bur cold area de losa, isolated from sputum samples of cystic fibrosis patients. The different banding patterns reflect the different numbers of O antigen, repeating units attached to the core oligosaccharides. A similar number of repeating units can be seen in samples one and six.
Don't forget that working with phenol dathyl ether can be hazardous in precautions such as wearing protective, personal protective equipment such as a lab coat, gloves, and goggles should be taken, and these experiments should be performed in a fume hood while attempting this procedure. It's important to perform the extractions quickly so you don't lose the samples during the processing. Following this procedure, Western immuno blotting or methods to visualize the lipo polysaccharide by silver STR or other methods need to be taken in order to visualize the relatedness between strains.
