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Lipomannan
Reagent:
Lipomannan, LM
Default Quantity:
100 µg
Production system:
The bacilli are grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, washed with PBS pH 7.4 and inactivated by gamma-irradiation. The cells are delipidated, then suspended in a PBS buffer containing 4% Triton X-114 and broken by French Press. This lysate is rocked at 4°C for 18 hours and insoluble material is removed by repeated centrifugation at 27,000 xg. The Triton X-114 extract is collected, heated at 37°C to allow biphasic partitioning and centrifuged at 27,000 xg. The detergent layer is collected and macromolecules including LM are recovered by ethanol precipitation. The ethanol insoluble material is suspended in PBS, and the proteins are digested and dialyzed out. The crude carbohydrate mixture is fractionated by size exclusion chromatography and the pure LM pooled. Buffer contaminants are removed by extensive dialysis. The solution is lyophilized and stored dry at -80°C.
Notes:
QC includes SDS-PAGE, Western blotting, LAL assay, NMR, and neutral sugar GC analysis. Contaminating LPS is avoided as all buffers and water used are endotoxin free. LAM from other M. tuberculosis strains and other Mycobacterium spp. will be made available for specific research needs.
References:
Chatterjee D., et al. J. Biol. Chem. 267:6228, 1992.
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