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Lipoarabinomannan
Reagent:
Lipoarabinomannan, LAM
Default Quantity:
500 µg
Strains available:
ManLAM from M. tuberculosis H37Rv, and smegLAM from M. smegmatis
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 LAM 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 LAM pooled. Buffer contaminants are removed by extensive dialysis. The solution is lyophilized and stored dry at -80°C.
Notes:
LAM is a cell wall product possessing many biological activities including immunogenicity, induction of TNF and the release of other cytokines, and inhibition of antigen processing. The nonreducing termini of H37Rv LAM are extensively capped with mannose. 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.
References:
Chatterjee D., et al. J. Biol. Chem. 267:6234, 1992.
Chatterjee D., et al. J. Biol. Chem. 267:6228, 1992.
Khoo K.-H., et al. J. Biol. Chem. 271:28682, 1996.
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