Isolation and Characterization of Lipopolysaccharides - Bacterial Pathogenesis: Methods and Protocols - page 9

3.6. Isolation of Lipid A (24)
Isolation of lipid A takes advantage of the labile linkage between the lipid
A backbone and the ketodeoxyoctanoate (KDO) in the LPS core. Mild acid
hydrolysis and heat are sufficient to disrupt this linkage. The lipid A is insoluble
in water and forms a precipitate that can be readily extracted by centrifugation.
Three methods are given in this section. The first is the classical method, which
has been most widely used. The second method allows extraction of small
amounts of relatively pure lipid A directly from whole cells without a prior LPS
extraction. The third method uses a relatively mild hydrolysis step to preserve
acid labile phosphorylation sites and head groups on the lipid A backbone. This
method is particularly useful in isolating lipid A for structural analysis.
3.6.1. Acetic Acid Hydrolysis Method
1. Lipid A can be isolated by mild acid hydrolysis of the purified LPS. Solubilize
LPS or LOS in aqueous 0.02% triethylamine and add acetic acid to a final
concentration of 1.5% (v/v).
2. Heat the mixture for2hat100°Candthen cool. Quantitatively precipitate lipid
A by adding 1 M HCl to a final pH of 1.5.
3. Centrifuge insoluble lipid A at 2000 × g , wash three times with cold distilled
water, and lyophilize.
3.6.2. Microlipid A Extraction from Whole Cells (25)
1. Suspend lyophilized crude or freshly washed bacterial cells (10 mg) in 400 μL of
isobutyric acid-ammonium hydroxide 1 M (5:3 v/v) and keep for2hat100°C
in a screw-cap test tube under magnetic stirring.
2. Cool mixture in ice water and centrifuge at 2000 × g for 15 min. Dilute supernatant
with water (1:1 v/v) and lyophilize.
3. Wash the sample twice with 400 μL of methanol and centrifuge at 2000 × g for
15 min.
4. Soluble insoluble lipid A and extract once in 100-200 μL of a mixture of
chloroform-methanol-water (3:1.5:0.25 v/v). For 1 mg samples, 100 μL of the
different solvent mixtures are used at each step (25) .
3.6.3. Lipid A Isolation (26)
1. Dissolve 5 mg of LPS in 500 μL of 10 m M sodium acetate (pH adjusted to 4.5
with 4 M HCl) containing 1% SDS and then place in an ultrasound bath until the
sample is dissolved.
2. Heat sample at 100 °C for 1 h. Dry the mixture by Speed Vac and remove SDS
by washing the sample with 100 μL of distilled water and 500 μL of acidified
ethanol (prepared by combining 100 μL of 4 M HCl with 20 mL of 95% ethanol)
followed by centrifugation at 2000 × g for 10 min.