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Dobos Lab: Lipids & Carbohydrates

Total Lipid

Reagent: Total Lipid
Default Quantity: 5 mg
Strains Available: H37Rv, HN878, and CDC 1551
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, washed with PBS pH 7.4, inactivated by gamma-irradiation and dried. The cellular lipids are extracted with 30 ml of chloroform/methanol (2:1) per gram of cells at 55°C for 18 hr. Cells are removed by filtration and contaminating hydrophilic molecules are removed by biphasic partitioning with water (Folch wash). The organic phase of the folch wash is collected, dried and stored at -80°C.
Notes: Total cellular lipids include those with known biological activities such as, trehalose dimycolate (TDM), and sulpholidids. Purification of individual lipids is done on a collaborative basis.
References:
Minnikin D.E., In Bacterial Cell Surface Techniques (I.C. Hancock and I.R. Paxton, Eds.) John Wiely & Sons, New York. pp 125-135, 1988.


Phosphatidylinositol Mannoside 1 & 2 (PIM1,2)

Reagent: Phosphatidylinositol mannoside, PIM1,2
Default Quantity: 0.5 mg
Production System: Cells are extracted with either chloroform/methanol/water (10:10:3). The organic soluble fraction is dried and titrated with cold acetone. The acetone insoluble fraction is applied to preparative thin-layer chromatography plates (20cm, Silica gel 60 F254, 1mm) in a solvent system of chloroform/methanol/water (60:30:6). PIMs are extracted from the silica plates and released from the dried matrix using 40% methanol in chloroform.
Notes: No structural differences between the PIMs of M. tuberculosis and M. bovis are known.
References:
Brennan, P., and Ballou, C.E., J. Biol. Chem. 242:3046, 1967.
Khoo, K.-H., et al. Glycobiol. 5:117, 1995.


Phosphatidylinositol Mannoside 6 (PIM6)

Reagent: Phosphatidylinositol mannoside, PIM6
Default Quantity: 250 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 PIM6 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 PIM6 pooled. Buffer contaminants are removed by extensive dialysis. The solution is lyophilized and stored dry at -80°C.
Notes: QC includes GC, LAL, analysis by MALDI, and TLC. No structural differences between the PIMs of M. tuberculosis and M. bovis are known.
References:
Brennan, P., and Ballou, C.E., J. Biol. Chem. 242:3046, 1967.
Khoo, K.-H., et al. Glycobiol. 5:117, 1995.


Mycolylarabinogalactan Peptidoglycan (mAGP)

Reagent: Mycolylarabinogalactan Peptidoglycan Complex, mAGP, Cell Wall Core
Default Quantity: 5 mg
Production System: Each strain is 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 bacilli are suspended (2 g/ml) in PBS containing DNase, and RNase, and broken in a French Press pressure cell at 4°C. Unbroken cells are removed by low speed (3,000 xg) centrifugation. The cell wall is isolated by centrifugation at 27,000 xg for one hour , the pellet suspended in PBS with 2% SDS and the cell wall associated proteins and lipoglycans are extracted at room temperature. Residual contaminating proteins are removed from the cell wall by further SDS extraction and proteinase-K treatment at 55°C, followed by several extractions with boiling SDS. The resulting mAGP is washed several times with water, and residual SDS is removed by extraction with large volumes of acetone.
Notes: The individual products that constitute the cell wall core (arabinogalatan, mycolic acid and peptidoglycan) are also available for specific research needs. QC of the mAGP includes quantification of protein and SDS contamination.
References:
Daffe M., et al. J. Biol. Chem. 265:6734, 1990.


Arabinogalactan (AG)

Reagent: Arabinogalactan, AG
Default Quantity: 1 mg
Production System: The mAGP of M. tuberculosis is hydrolyzed with KOH in methanol. The insoluble material (AGP) is collected and the arabinogalactan (AG) is released from the peptidoglycan (PG) by mild acid hydrolysis with H2SO4. The soluble AG is separated from the insoluble PG by centrifugation. Soluble AG is neutralized, dialyzed against H2O and dried.
Notes: QC includes carbohydrate analysis by GC-MS.
References:
Daffe M., et al. J. Biol. Chem. 265:6734, 1990.


Peptidoglycan (PG)

Reagent: Peptidoglycan, PG
Default Quantity: 0.5 mg
Production System: The mAGP of M. tuberculosis is hydrolyzed with KOH in methanol. The insoluble material (AGP) is collected and the arabinogalactan (AG) is released from the peptidoglycan (PG) by mild acid hydrolysis with H2SO4. The soluble AG is separated from the insoluble PG by centrifugation. PG pellets are resuspended H2O, aliquotted and dried.
Notes: QC includes carbohydrate analysis by GC-MS and amino acid analysis.
References:
Daffe M., et al. J. Biol. Chem. 265:6734, 1990.


Mycolic Acid Methyl Esters (MAME)

Reagent: Mycolic acid methyl esters, MAME
Default Quantity: 1 mg
Production System: Mycolic acids are released from the mAGP of M. tuberculosis by alkaline hydrolysis with KOH in methanol. The soluble mycolic acids are collected, and neutralized. Chloroform and water are added to the mycolic acid suspension to form a biphasic partition. The organic fraction containing mycolic acid is collected and dried.
Notes: QC performed by TLC.


Lipoarabinomannan (LAM)

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.


Lipomannan (LM)

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.


Sulfolipid-1 (SL-1)

Reagent: Sulfolipid-1, SL-1
Default Quantity: 0.25 mg
Production System: Sulfolipid-1 (SL-1) is first purified by extracting H37Rv Mtb cells with 2:1 chloroform/methanol and loading onto a silica gel column. The column is washed with chloroform, then eluted with 5% methanol in chloroform. This fraction, enriched in TDM and SL-1, is loaded onto C18 reverse phase SepPak filters and eluting with 60% chloroform in methanol to remove the TDM, then 25% to elute the SL-1.
Notes: For most requests, this product will be made on demand due to yield and stability issues. Therefore, a delay should be anticipated.
Sulfolipid appears to be very labile. Dry storage at -80°C is recommended to prevent breakdown. As little as two days storage in solution at 4°C causes SL-I to become very non-polar, as evidenced by running on 2D-TLC.


Trehelose Dimycolate (TDM)

Reagent: Trehelose Dimycolate, TDM
Default Quantity: 0.25 mg
Production System: Trehalose dimycolate (TDM) is purified by extracting H37Rv Mtb cells with 2:1 chloroform/methanol and loading onto a silica gel column. The column is washed with chloroform, then eluted with 5% methanol in chloroform. TDM, enriched in this fraction, is further purified by loading onto C18 reverse phase SepPak filter and eluting with 60% chloroform in methanol.
Notes: For most requests, this product will be made on demand; therefore, a delay should be anticipated

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