Lipid A Preparation

Lipid A is prepared using a modification of the method of Rietschel et al (1977).

Equimolar amounts (standardized for KDO content) of lyophilized LPS is resuspended in 15ml in 50mM sodium acetate buffer, pH 3.0. The buffer is at pH 3.0 in an attempt to lessen the chance of removing labile phosphate groups from Lipid A.The resuspended LPS is heated at 100^oC for 90 minutes, then frozen and thawed four times to promote aggregation of Lipid A.

Samples are centrifuged in thick-walled glass tubes at 9500 rpm for 5 minutes, washed once with sodium acetate buffer and washed twice more with deionized water to remove sodium acetate prior to lyophilization.

Samples are resuspended in distilled H₂O and assayed for the loss of KDO content to ensure the complete removal of LPS carbohydrate from Lipid A.