BEI Resources

Subcellular fractions are available for the standard laboratory strain H37Rv as well as clinical isolates CDC1551 and HN878.

Live Whole Cells

Reagent: Live Mycobacterium tuberculosis Whole Cells.
Quantities available: 1ml frozen stocks
Strains Available: H37Rv, CDC1551, and HN878
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium. For frozen stocks, glycerol is added to a final concentration of 20% and 1 ml aliquots are frozen at -80°C.
Notes: Live cells of M. tuberculosis are supplied only to laboratories that provide a completed BSL-3 Certification Letter.

Irradiated Whole Cells

Reagent: Inactivated Mycobacterium tuberculosis Whole Cells, γ-irradiated whole cells.
Quantities available: 10 grams
Strains Available: H37Rv, CDC1551, and HN878
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium. The bacilli are harvested and frozen at -80°C. For inactivation, the cell pellet is exposed to 2.4 megaRads of ionizing gamma irradiation using a 137Cs source. Confirmation of inactivation is performed by Alamar Blue Assay. The inactivated cell pellets are stored at -80°C.
Notes: A dose of 2.4 mRads of gamma irradiation kills M. tuberculosis to a 1020 degree of certainty while maintaining 93-95% of the biological activity of the enzymes.

Whole Cell Lysate

Reagent: Whole Cell Lysate, WCL.
Quantities available: 10 mg
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and inactivated by gamma-irradiation. Cells are suspended (2 g/ml) in PBS buffer containing 8 mM EDTA, proteinase inhibitors, DNase and RNase. Cells are disrupted by French Press until ~ 90% breakage is obtained (monitored by acid fast staining). The lysate is centrifuged at 3,000 x g to pellet unbroken cells, and the supernatant isolated. The protein content is quantified using the BCA protein assay, and aliquots are stored at -80°C.
Notes: This crude preparation contains proteins, lipids, and carbohydrates present within the bacterial cell, including cell wall, cytosol, and membrane.

Culture Filtrate Proteins

Reagent: Mixed Culture Filtrate Proteins, CFP.
Quantities available: 1 mg
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium. The culture supernatant is harvested from the live cells by passing through a 0.2 micron filter. The CFP is concentrated by Amicon ultrafiltration using a membrane with a molecular weight cutoff of 5,000 Da. The concentrated material is dialyzed against 0.01 M ammonium bicarbonate, quantitated with the BCA protein assay, aliquoted, lyophilized, and stored at -80°C.
Notes: This preparation includes most of the excreted/secreted proteins of M. tuberculosis. Individual lots are subjected to quality control procedures to ensure uniformity and lack of bacterial contamination.
References:
Dobos, K.M. et al., Infect. Immun. 63:2846, 1995.
Sonnenberg, M.G. et al., Infect. Immun. 65:4515, 1997.

Cell Wall Fraction

Reagent: Cell Wall Fraction, CW.
Quantities available: 1 mg
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and inactivated by gamma-irradiation. The bacilli are suspended (2 g/ml) in PBS containing 8 mM EDTA, DNase, RNase and a proteinase inhibitor tablet, and broken in a French Press pressure cell at 4°C. Unbroken cells are removed by low speed (3,000 x g) centrifugation. The cell wall is isolated by centrifugation at 27,000 x g for one hour and washed 2 times in PBS. The final cell wall pellet is suspended and dialyzed in 0.01M ammonium bicarbonate, quantified by BCA protein assay for protein content, and stored at -80°C.
Notes: This preparation contains proteins and non-protein compounds such as mAGP.
References:
Hirschfield, G.R. et al. J. Bacteriol. 172:1005, 1990.

Cell Membrane Fraction

Reagent: Cell Membrane Fraction, MEM.
Quantities available: 1 mg
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and inactivated by gamma-irradiation. The bacilli are suspended (2 g/ml) in PBS containing 8 mM EDTA, DNase, RNase and a proteinase inhibitor tablet, and broken in a French Press pressure cell at 4°C. Unbroken cells are removed by low speed (3,000 x g) centrifugation. The cell wall is removed by centrifuging twice at 27,000 x g. The supernatant is subjected to a 100,000 x g centrifugation for four hours. The resulting membrane pellet is washed with PBS, then suspended and dialyzed in 0.01 M ammonium bicarbonate. Protein content is determined using the BCA protein assay and the membrane is stored at -80°C.
Notes: This preparation contains cytoplasmic membrane and components of the outer lipid layer of M. tuberculosis.
References: Lee, B.-Y., et al. Infect. Immun. 60:2066, 1992.

Cytosol Fraction

Reagent: Cytosol Fraction, CYT.
Quantities available: 1 mg
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and inactivated by gamma-irradiation. The bacilli are suspended (2 g/ml) in PBS containing 8 mM EDTA, DNase, RNase and a proteinase inhibitor tablet, and broken in a French Press pressure cell at 4°C. Unbroken cells are removed by low speed (3,000 x g) centrifugation. The cell wall is removed by centrifuging twice at 27,000 x g. The supernatant is subjected to a 100,000 x g centrifugation for four hours. The supernatant is collected, dialyzed against 0.01 M ammonium bicarbonate, and the protein content is determined using the BCA protein assay. This material is stored at -80°C.
Notes: In addition to proteins of the cytosol this preparation will contain soluble material released from the cell wall during disruption of the bacilli.
References:
Lee, B.-Y., et al. Infect. Immun. 60:2066, 1992.
Hirschfield, G.R., et al. J. Bacteriol. 172:1005, 1990.

Only Available for H37Rv

SDS – Soluble Cell Wall Proteins

Reagent: SDS-Soluble Cell Wall Proteins, SCWP.
Quantities available: 1 mg
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and inactivated by gamma-irradiation. Cell walls are isolated as described (see cell wall fraction for details). The cell wall pellet is extracted with 2% SDS in PBS at room temperature for two hours. The SDS extract of the cell wall is removed by centrifugation and SDS is removed by a paired-ion extraction. The protein content is quantified using the BCA protein assay and aliquots are stored at -80°C.
Notes: Minimal SDS concentrations are achieved using a paired-ion exchange and the amount of residual SDS contamination is determined.
References:
Hirschfield, G.R., et al. J. Bacteriol. 172:1005, 1990.

TX-114 – Soluble Protein Pool

Reagent: TX-114 Soluble Protein Pool.
Quantities available: 1 mg
Production system: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and inactivated by gamma-irradiation. Cells are broken by French press in 4% triton X-114. The materials is then partitioned at 37°C and centrifuged at 27,000 xg, 25°C. The upper aqueous layer is removed and the triton layer is extracted twice more in the same way. The final triton layer is acetone precipitated to remove the triton. The pellet from the acetone precipitation is phenol extracted, then dialyzed into water. The protein content is quantified using the BCA protein assay and aliquots are stored at -80°C.
Notes: Larger quantities are available for laboratories needing to purify products from this preparation or for laboratories to use for TLR2 signaling.
References:
Radolf, J.D., et. al. Infect. Immun. 56:490-8.

Native proteins are available in default quantities and are not always stocked. Native proteins are purified from M. tuberculosis strain H37Rv. The QC for these products consists of SDS-PAGE and western blot analysis.

Antigen 85

Reagent: Antigen 85 Complex (Mixture of Rv3804c (Ag85A, FbpA), Rv1886c (Ag85B, FbpB), and Rv0129c (Ag85C, FbpC)
Default Quantity: 500 µg
Production System: CFPs are precipitated with a 40% saturated solution of ammonium sulfate. The precipitate is suspended and applied to phenyl sepharose HPLC. Antigen 85 is obtained by increasing the pH and eluting with a high concentration of ethylene glycol. If necessary, additional purification is performed using size exclusion chromatography. The purified product is dialyzed, quantified, aliquoted, and lyophilized. Aliquots are stored at -80°C.
Notes: The Ag85 complex contains three gene products of 30-31 kDa. The individual components of the Ag85 complex (Ag85A, Ag85B and Ag85C) are available for specific research needs, and the default quantity for these is 100 µg.
References:
Wiker H.G. and Harboe M., Microbiol. Rev. 56:648, 1992.

45kDa Glycoprotein

Reagent: Rv1860, 45 kDa glycoprotein, MPT32, ModD, Apa
Default Quantity: 500 µg
Production System: CFPs are precipitated with a 40% saturated solution of ammonium sulfate. The precipitate is suspended and incubated with Conconavalin-A resin, and the 45 kDa is eluted with alpha-methyl mannoside. A final purification is performed using hydrophobic interaction (phenyl sepharose) HPLC. The purified product is dialyzed, quantified, aliquoted, and lyophilized. Aliquots are stored at -80°C.
Notes: The 45 kDa glycoprotein appears as a doublet by SDS-PAGE. The major component is 45 kDa and the minor component is a 42 kDa degradation product. The mature MPT 32 glycoprotein has a calculated molecular weight of 30.2 kDa, however the abundance of proline residues along with glycosylation causes this molecule to migrate at 45 kDa by SDS-PAGE.
References:
Dobos K.M. et al., Infect. Immun. 63:2846, 1995.
Dobos K.M. et al., J. Bact. 178:2498, 1996.

GroES

Reagent: Rv3418c, GroES, 14 kDa heat shock protein
Default Quantity: 500 µg
Production System: Supernatant from a 40% saturated ammonium sulfate precipitation of CFPs is precipitated with 50% saturated ammonium sulfate. The pellet is suspended, and run over a conA column. The material that does not bind to the column is then resolved by reversed phase HPLC column. Fractions containing the GroES homologue are pooled. The purified product is dialyzed, quantified, aliquoted, and lyophilized. Aliquots are stored at -80°C.

Alpha-crystallin

Reagent: Rv2031c, alpha-crystallin, 16 kDa antigen, HspX
Default Quantity: 100 µg
Production System: Whole cell lysate is extracted with 0.1% n-octylthioglucoside in PBS and centrifuged at 27,000 xg for one hour. The supernatant is fractionated by isoelectric focusing (BioRad Rotofor system). Fractions containing 16kDa are pooled and cleaned up using size exclusion chromatography and pure fractions of 16 kDa are pooled. The purified product is dialyzed, quantified, aliquoted, and lyophilized. Aliquots are stored at -80°C.
References:
Lee B., et al. Infect. Immun. 60:2066, 1992.

PhoS1

Reagent: Rv0934, PhoS1, 38 kDa antigen, Anitgen 5, PstS1
Default Quantity: 100 µg
Production System: The proteins soluble in 40% ammonium sulfate are precipitated with a 70% saturated ammonium sulfate solution. These proteins are suspended in ConA binding buffer and applied to a ConA column. ConA elution buffer, containing alpha-D-mannopyranoside, is used to elute the bound proteins. The bound material is applied to a phenyl sepharose column and eluted using a gradient of decreasing ammonium sulfate. Fractions containing pure 38kDa are pooled. The purified product is dialyzed, quantified, aliquoted, and lyophilized. Aliquots are stored at -80°C.
Notes: It should be noted that the cell wall associated and culture filtrate forms of PhoS1 seem to differ in their hydrophobic characteristics. However, we have not yet determined if there is a chemical difference between the cell wall and culture filtrate forms of the PhoS1 homologue. The PhoS1 provided by this contract is purified from culture filtrate.

Mpt64

Reagent: Rv1980c, Mpt64
Default quantity: 100 µg
Production System: Culture filtrate proteins precipitated with a solution of 50% saturated ammonium sulfate are resuspended in phosphate buffered saline solution for antibody affinity purification. Monoclonal anti-Mpt64 antibodies are harvested and purified from hybridomas and covalently bound to resin beads for column purification. CFPs are passed over the antibody column and eluted after washing to purify. The purified product is dialyzed, quantified, aliquoted, and lyophilized. Aliquots are stored at -80°C.

1411c

Reagent: Rv1411c, lprG
Default quantity: 100 µg
Production System: Culture filtrate proteins precipitated with a solution of 70% saturated ammonium sulfate are resuspended in phosphate buffered saline solution for antibody affinity purification. Monoclonal anti-Rv1411c antibodies are harvested and purified from hybridomas and covalently bound to resin beads for column purification. CFPs are passed over the antibody column and eluted after washing to purify. The purified product is dialyzed, quantified, aliquoted, and lyophilized. Aliquots are stored at -80°C.

Recombinant expression vectors containing Mycobacterium tuberculosis genes and protocols for generation of recombinant proteins.

Reference Standards

The production of recombinant proteins for routine use in laboratory studies is now enabled via ordering the recombinant clone for the antigen of interest. Detailed protocols and references are provided with orders so that laboratories can make proteins as needed without the delays of shipping and TBVTRM prioritization. The TBVTRM Contract will continue to generate the following recombinant proteins: ESAT-6, CFP10, Ag85a and Ag85b. These can be ordered without justification and are made available for use as reference standards. These standards may change as the needs of the research community change.

Large Scale Studies

The production of recombinant proteins for routine use in laboratory studies is now enabled via ordering the recombinant clone for the antigen of interest. Detailed protocols and references are provided with orders so that laboratories can make proteins as needed without the delays of shipping and TBVTRM prioritization. The use of recombinant proteins for in vitro studies as part of a large clinical study or trial and for structural analysis demands a purification scale that is often incompatible with the resources of most academic institutions. Thus, recombinant proteins will be made available to laboratories involved in large scale clinical trials and structural studies. Principal investigators who wish to be considered must first register with the contract and have a fully executed MTA on file. These needs will then be addressed via submission of a detailed research plan and justification. All submissions will be considered, though there may be a significant delay based on the scope of project and prior proposals in queue. Additionally, all activities in this scope of work will be considered collaborations; thus there must be a guarantee of disclosure by all parties. Please contact Dr. Karen Dobos for additional information or to submit a research proposal.

Reagent: Recombinant expression vectors containing Mycobacterium tuberculosis genes and protocols for generation of recombinant proteins.
Default Quantity: 1 µg plasmid DNA
Production System: All genes are amplified by PCR and cloned into pET23b or pET15b (Novagen) for expression in E. coli or pVV16 (a derivative of pMV261 allowing His-tag fusion) for expression in M. smegmatis. All proteins are His-tagged.
Notes: Recombinant proteins will only be provided as reference standards or for large scale studies (see below for more details). All other laboratories will receive plasmid DNA and detailed protocols for gene expression and protein purification.
Plasmids listed are expressed in E. coli unless otherwise stated. In general, plasmids expressed in E. coli are cloned without a signal sequence and those expressed in M. smegmatis are cloned with signal sequence (unless otherwise noted).

* Approximate yields are given if known, based on recovery of purified protein from a 1L culture.
Note: This list is updated on a continual basis as new recombinant products are generated.

All lipids are carbohydrates are purified from M. tuberculosis strain H37Rv, unless otherwise stated.

Total Lipid

Reagent: Total Lipid
Default Quantity: 5 mg or as indicated below
Strains Available: H37Rv, HN878, and CDC 1551, M. leprae (500 µg), M. bovis
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, 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 sulpholipids. 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 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 and inactivated by gamma-irradiation. The cells are delipidated, then suspended in a PBS buffer containing 8% 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 endotoxin free water, 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 SDS-PAGE, Western Blotting, EndoZyme II Assay, GC, and analysis by MALDI. 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 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: As indicated below
Strains Available: ManLAM from M. tuberculosis H37Rv (500 µg), and lepLAM from M. leprae (100 µg)
Production System: The bacilli are grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and inactivated by gamma-irradiation. The cells are delipidated, then suspended in a PBS buffer containing 8% 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 endotoxin free water, 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, EndoZyme II 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 and inactivated by gamma-irradiation. The cells are delipidated, then suspended in a PBS buffer containing 8% 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 endotoxin free water, 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, EndoZyme II Assay, NMR, and neutral sugar GC analysis. Contaminating LPS is avoided as all buffers and water used are endotoxin free. LM 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

Acetone Soluble and Insoluble Lipids

Reagent: Acetone Soluble and Insoluble Lipids
Default Quantity: 0.5 mg
Strains Available: H37Rv
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, inactivated by gamma-irradiation and dried. Total lipid is resuspended in chloroform/methanol. Acetone soluble and insoluble lipids are then separated by centrifugation and dried.

Neoglycoproteins (ND-O-BSA and -HSA)

Reagent: ND-O-BSA and -HSA (SYNTHETIC PGL-I)
Default Quantity: 0.25 mg
Production System: (Natural Disaccharide O-linked to Bovine Serum Albumin (ND-O-BSA) and -Human Bovine Albumin (HSA) are synthetic products developed based on Mycobacterium leprae antigenicity. The synthesis is performed by the conjugation of the disaccharide hydrazide segment of phenolic glycolipid I of M. leprae with albumin (either bovine or human origin), via acyl-azide intermediate. A stirred solution of the disaccharide hydrazide is converted in an unstable acyl-azide product in a time-dependent reaction under freezing conditions. This acyl-azide is used for conjugation with a previously prepared BSA (or HSA) solution (pH 9.0 to 9.2) under cold conditions overnight. The final conjugated product is then subjected to buffer exchange/dialysis and HPLC-purification to remove unbound albumin.

Phthiocerol Dimycocerosate (PDIM)

Reagent: PDIM
Default Quantity: 0.5 mg
Strains Available: H37Rv
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, inactivated by gamma-irradiation and dried. Total lipid is applied to TLC plates. Specific bands are selected, removed, and purified. The process is repeated for further purification.

Mycobactin

Reagent: PDIM
Default Quantity: 0.1 mg
Strains Available: H37Rv
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, inactivated by gamma-irradiation and dried. Total lipid is extracted with ethanol and applied to preparative TLC plates. Mycobactin-specific bands are removed from the plate and extracted with chloroform/methanol. After centrifugation, extracts are dried.

Phenolic Glycolipid (PGL-1)

Reagent: PGL-1
Default Quantity: 1 mg
Strains Available: M. canetti
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, inactivated by gamma-irradiation and dried. Total lipid is applied to preparative TLC plates. PGL-specific bands are removed and extracted with chloroform/methanol, and dried.

Trehalose Monomycolate (TMM)

Reagent: TMM
Default Quantity: 0.25 mg
Strains Available: H37Rv
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, inactivated by gamma-irradiation and dried. Enriched total lipid is applied to preparative TLC plates. TMM-specific bands are removed and extracted with chloroform/methanol. After centrifugation and filtering, the extracts are dried.

6-O-Methylglucose-Containing Lipopolysaccharides (MGLP)

Reagent: MGLP
Default Quantity: 0.1 mg
Strains Available: H37Rv
Production System: Each strain is grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium, inactivated by gamma-irradiation and dried. Cells are delipidated with 10:10:3. After centrifugation and filtering, the extracts are dried and resuspended in MilliQ water. Total lipid extract is run through a centrifugal filter unit. After drying, product is resuspended in water and eluted through a Sep-Pak C18 cartridge. Fractions are dried and then resuspended in water.

Genomic DNA is available in the following strains: H37Rv and Clinical isolate CSU93 (KY/TN 95-031551, CDC 1551), M.BOVIS TB17-02518 (25 µg), M.BOVIS 95-1315 (80 µg), M. LEPRAE NHDP-63 (2 µg), M. LEPRAE THAI-53 (2 µg), M. LEPRAE STRAIN BR4923(2 µg)

Reagent: Genomic DNA
Default Quantity: 100 µg (unless otherwise stated above)
Strains Available: H37Rv and Clinical isolate CSU93 (KY/TN 95-031551, CDC 1551), M.bovis TB17-02518 (25 µg), M.bovis 95-1315 (80 µg), M. leprae NHDP-63 (2 µg), M. leprae Thai-53 (2 µg), M. leprae Strain Br4923(2 µg)
Production System: The bacilli are grown to late-log phase (day 14) in glycerol-alanine-salts (GAS). Cell lipids are removed by chloroform/methanol (2:1) extraction in an equal volume of Tris-EDTA buffer. Delipidated cells are exposed to lysozyme and RNase overnight followed by SDS and Proteinase K treatment. Genomic DNA is isolated from contaminating proteins using organic extraction. To remove polysaccharide contaminants, DNA is precipitated with isopropanol and stored at 4°C as a dry pellet.
Notes: Quality control for this reagent includes agarose gel electrophoresis and spectrophotometric analysis.
References:
Belisle, J.T., and Sonnenberg, M.G., Methods Mol. Biol. 101:31, 1998.

Note: Monoclonal antibodies are provided as culture supernatents. Polyclonal antibodies are provided as sera. The default amount of each antibody is based on its working titer.

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M001.2: Preparation of GAS medium

M002.3: Preparation of GAS medium with tween

M003.1: Preparation of Sauton’s medium

M004.1: Preparation of Sauton’s medium with tween

M005.2: Preparation of 7H9 medium

M006.2: Preparation of 7H9 medium with tween

M007.0: Preparation of 7H9-Dextrose medium

M008.0: Preparation of 7H9-Dextrose medium with tween

M009.3: Preparation of 7H11 agar plates

M010.1: Preparation of 7H11-Dextrose agar plates

M011.3: Preparation of Nutrient agar plates

M013.0: Preparation of Mycobactin HR Media and Plates

MO14.2: Preparation of SPAS medium

MO15.2: Preparation of SPAS medium with tween

MO16.2: Preparation of Middlebrook 7H9 broth with sodium pyruvate

MO17.2: Preparation of Middlebrook 7H9 broth with tween and sodium pyruvate

MO18.2: Preparation of Middlebrook 7H11 agar with sodium pyruvate

MO19.2: Preparation of Middlebrook 7H10 agar with sodium pyruvate

MO20.1: Preparation of Proskauer and Beck minimal media

MO21.0: Preparation of Complete DMEM Media for Hybridoma Growth

MO22.0: Preparation of Complete DMEM for Growth of J774a.1 Cells

MO23.0: Preparation of Complete RPMI for Growth of THP-1 Cells

MO24.0: Preparation of Exosome-Free FBS

MO25.0: Preparation of Middlebrook 7H9 broth protocol for Lys and Pan Auxotroph

MO26.0: Preparation of Middlebrook 7h11 Agar protocol for Ls and Pan Auxotroph

MO27.2: Preparation of Complete DMEM/F12 Media for Hybridoma Growth

MO28.3: Preparation of Complete IMDM Media for Hybridoma Cell Growth

MO29.0: Preparation of Complete RPMI Media for Hybridoma Cell Growth

MO30.0: Preparation of Middlebrook 7H9 broth with tween and glycerol

MO31.0: Preparation of GAS with glycerol

MO32.0: Preparation of GAS medium +0.05% Tween 80 protocol +20% glycerol

R001: Preparation of 32% Triton

R002: Preparation of 1st fix for gels

R003: Preparation of 2nd fix for gels

R010.1: Preparation of western blot transfer buffer

R011: Preparation of charring spray

R012: Preparation of α-napthol spray

R013: Preparation of Dittmer-Lester spray

R014: Preparation of TAE Buffer

PP001.3: Establishment of frozen stocks of Mycobacterium tuberculosis (small scale)

PP003.4: Large-scale growth of Mycobacterium tuberculosis

PP004.2: Gamma-irradiation of M. tuberculosis

PP006.4: Preparation of CFPs using 2L Stirred Cell

PP007.4: Preparation of whole cell lysate

PP008.4: Preparation of subcellular fractions

PP009.3: Preparation of genomic DNA

PP010.3: Preparation of TX-114 proteins

PP011.2: Preparation of mAGP

PP012.2: Preparation of Arabinogalactan

PP013.2: Preparation of peptidoglycan

PP014.0: Preparation of mycolic acids

PP015.6: Preparation of LAM, LM, and PIM (LLP)

PP016.7: Separation of LAM, LM, and PIM

PP017.3: QC of LAM, LM, and PIM6

PP018.2: Isolation of total lipid

PP019.4: Purification of Con-A reactive CFPs

PP020.4: Preparation of Ag85 complex

PP021.6: Preparation of Ag85 A, B, and C

PP022.6: Purification of MPT32

PP023.6: Purification of the 16kDa ACR/HspX

PP024.9: Purification of PhoS1 (38kDa protein) from the CFP

PP025.1: Preparation of SDS soluble cell wall protein

PP026.5: Preparation of PIM

PP027.0: Purification of SapM

PP028.2: LAM Removal from hydrophilic subcellular fractions

PP029.4: Purification of TDM and SL

PP030.2: Purification of TMM

PP031.2: Purification of 19kDa

PP032.2: Purification of Mycobactin

PP034.0: Preparation of Sulfolipid-1

PP035.5: Purification of native GroES

PP036.1: Preparation of Acetone-Soluble and Insoluble Total Lipid 

PP037.3: Purification of PDIM

PP038.0: Purification of Phenolic Glycolipid from M. canetti

PP039.1: LAM Removal from hydrophobic/mixed subcellular fractions​

PP0040.2: Growth of Mycobacterium Normoxic/Hypoxic Culture Pairs Using a Fermentor/Bioreactor

PP041.2: Large-scale growth of Mycobacterium smegmatis

PP042.2: Large scale purification of Non-Mtb Mycobacterium spp genomic DNA 

PP049.1: Pellicle Growth of M. tuberculosis

PP050.1: Purification of M.leprae from armadillo tissue

PP051.0: Subcellular Fraction of M. leprae

PP052.0: Isolation of Native PGL-I from M. leprae

PP053.1: Ag85a,b,c MALDI-TOF/TOF MS Protocol

PP054.1: Establishment of frozen glycerol stocks of Mycobacterium smegmatis, small scale

PP055.0: Large-scale growth of Mycobacterium bovis

PP056.0: Mycobacterial Extracellular Vesicle (MEV) Enriched and Depleted CFP

PP057.0: Separation of Soluble Filtrates, including CFP, for Native Protein Starting Material

PP058.0: Total Lipid Enrichment

PP059.2: Demannosylated LAM

PP060.2: Purification of mGLP

PP061.1: Purification of Native Mpt64

PP062.1: Purification of Native Rv1411c

RP001: Generation of Recombinant Clone Frozen Stocks

RP002: Establishing Stocks of Low Copy Plasmids

RP003a: Production of Recombinant Proteins

RP003b: Production of Recombinant Proteins Denaturing Conditions

RP004: Production of ESAT6

RP005: Production of CFP10

RP006: Small- Scale Purification of Plasmid DNA

RP007: Production of rec GlcB

RP007msmeg: Production of rec GlcB from M. smegmatis

RP008: Production of rec Rv1411c

RP009: Production of Recombinant Psts1

RP010: Production of rec Ag85A

RP011: Production of rec Ag85B

RP012: Production of rec Ag85C

RP013: Production of Recombinant ModD 

RP014: Production of rec DnaK

RP015: Production of rec GroEL2

RP016: Production of rec Mpt64

RP017: Production of Recombinant TPX

RP018: Production of Recombinant Rpll

RP019: Production of rec BRFB

RP020: Production of rec Mpt51

RP021: Production of rec HspX

RP022: Production of rec Rv2626c

RP023: Production of rec Rv0577

RP025: Production of rec KatG

RP026: Production of rec Rv3763

RP027: Production of Recombinant OmpA Non-Denaturing Conditions

RP028: Production of GroES

RP029.3: Production of Recombinant Proteins using a Bioreactor

RP030: Production of Recombinant Rv1738 Non-Denaturing Conditions

AB100.6: Cell Line Maintenance

AB102.1: Antibody QC

AB103.7: Monoclonal Fusion

AB104.6: Screening and Subcloning

AB105.5: Freezing Cell Lines

AB106.1: Isotyping of Hybridoma Monoclonal Antibodies

AB107.1: Monoclonal Antibody (MAB) Production Using a Murine Model (Immunizations, Bleeds, and Blood Processing)

AB108.1: Purification of Monoclonal Antibody by Melon Gel