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Cells & Sub-cellular Fractions

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 by filtration washed with PBS pH 7.4, pelleted by centrifugation 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, washed with PBS pH 7.4., 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, washed with PBS pH 7.4 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, washed with PBS pH 7.4, 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, washed with PBS pH 7.4 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, washed with PBS pH 7.4., 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 running the extract over a column packed with Extracti gel (Pierce Chemical). The protein content is quantified using the BCA protein assay and aliquots are stored at -80°C.
Notes: Minimal SDS concentrations are achieved using Extracti-gel 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, washed with PBS pH 7.4, 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

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, Ag85, 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 against 0.01 M ammonium bicarbonate, lyophilized, and 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 250 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. This fraction is concentrated and dialyzed. A final purification is performed using hydrophobic interaction (phenyl sepharose) HPLC. The purified 45 kDa glycoprotein is lyophilized and 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: A supernatant from a 40% saturated ammonium sulfate precipitation of CFPs is precipitated with 70% 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. Aliquoted samples 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. Pure fractions of 16 kDa are pooled, dialyzed against 0.01 M ammonium bicarbonate, quantified using the BCA protein assay, lyophilized, and 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: 250 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 and dialyzed. 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

Recombinant Plasmids

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).

Table of recombinant plasmids available through the TB Vaccine Testing and Research Materials Contract
(Plasmids listed are expressed in E. coli unless otherwise stated)

Gene/Protein
Plasmid
Observed Protein Size
Expected Protein Yield*
Rv3763/19kDa/LpqH pMRLB51 19 kDa 0.5 mg
Rv2032/Acg pMRLB48 37 kDa
Rv0733/Adk pMRLB25 29 kDa
Rv3804/Ag85A pMRLB41 32 kDa 1 mg
Rv1886c/Ag85B pMRLB47 32 kDa 1 mg
Rv0129c/Ag85C pMRLB16 32 kDa 6 mg
Rv3841/BfrB pMRLB5 20 kDa 1 mg (high endotoxin)
0.1 mg (after ET removal)
Rv3841/BfrB pMRLB63
M. smegmatis
20 kDa
Rv3874/CFP10 pMRLB46 11 kDa 1 mg
Rv0350/DnaK pMRLB6 70 kDa 1 mg
Rv3875/Esat6 pMRLB7 10 kDa 5 mg
Rv3019c/EsxR pMRLB67 10 kDa
Rv1827/GarA pMRLB71 23 kDa
Rv1837c/GlcB pMRLB8 80 kDa 0.2 mg
Rv1837c/GlcB pMRLB45
M. smegmatis
80 kDa
Rv2220/GlnA pMRLB66 60 kDa
Rv0440/GroEL2 pMRLB1 65 kDa 0.5 mg
Rv3418c/GroES pMRLB9 10 kDa
Rv2031c/HspX pMRLB15 16 kDa 5 mg
Rv1484/InhA pMRLB10 29 kDa
Rv1908c/KatG pMRLB11 81 kDa 3 mg (not clean)
0.05 mg (after cleanup)
Rv3006/LppZ pMRLB54 39 kDa
Rv3006/Lppz pMRLB55
M. smegmatis
39 kDa
Rv1270c/LprA pMRLB50 25 kDa
Rv1411c/LprG pMRLB43 27 kDa 1.5 mg
Rv1411c/LprG pMRLB44
M. smegmatis
27 kDa
Rv1860/Mpt32 pMRLB17 45 kDa 0.1 mg
Rv3803c/Mpt51 pMRLB38 31 kDa 4 mg
Rv1926c/Mpt63 pMRLB69 20 kDa
Rv1980c/Mpt64 pMRLB12 25 kDa 3 mg
Rv0899/OmpA pMRLB42 33 kDa
Rv0934/PhoS1/PstS pMRLB2 38 kDa 0.75 mg
Rv0652/RplL pMRLB13 13 kDa 0.25 mg
Rv0379/SecE2 pMRLB56 8 kDa
Rv0379/SecE2 pMRLB57
M. smegmatis
8 kDa
Rv3846/SodA pMRLB68 25 kDa
Rv0432/SodC pMRLB60 24 kDa
Rv0432/SodC pMRLB61
M. smegmatis
24 kDa
Rv0432/SodC
(w/o signal sequence)
pMRLB62
M. smegmatis
24 kDa
Rv1932/Tpx pMRLB14 17 kDa 3 mg
Rv3914/TrxC/Mpt46 pMRLB58 12 kDa
Rv3914/TrxC/Mpt46 pMRLB59
M. smegmatis
12 kDa
Rv0164 pMRLB70 22 kDa
Rv0577 pMRLB24 27 kDa
Rv1738 pMRLB49 10 kDa
Rv1813c pMRLB53 15 kDa
Rv2626c pMRLB28 15 kDa 8 mg
Naked Plasmid pVV16

* 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.

Lipids and Carbohydrates

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

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

Genomic DNA

Genomic DNA is available in the following strains: H37Rv and Clinical isolate CSU93 (KY/TN 95-031551, CDC 1551)

Reagent: Genomic DNA
Default Quantity: 100 µg
Strains Available: H37Rv and Clinical isolate CSU93 (KY/TN 95-031551, CDC 1551)
Production System: The bacilli are grown to late-log phase (day 14) in glycerol-alanine-salts (GAS) medium and harvested by centrifugation. 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.

Antibodies

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.

Scroll down to view the entire table of available antibodies.

Specificity
Antibody Designation
(Original Designation)
Ab Type
Isotype
Titer
19kDa lipoprotein (Rv3763, LpqH) IT-12 (HYT6) Mouse Monoclonal IgG1 1:20
IT-19 (TB23) Mouse Monoclonal IgG1 1:40
16kDa (Rv2031c, HspX, α-crystallin, acr) IT-20 (TB68) Mouse Monoclonal IgG1 1:100
CS-49 Mouse Monoclonal IgG1 1:100
CS-50 Mouse Monoclonal IgA 1:100
Antigen 85 Complex (Rv3804c, FbpA, Rv1886c, FbpB, Rv0129c, FbpC) IT-44 (HBT7) Mouse Monoclonal IgG2a 1:50
IT-55 (HRT2)*
poor reactivity to Rv3804c
Mouse Monoclonal IgG2a 1:20
CS-90 Mouse Monoclonal IgM 1:20
α-Ag85 Rabbit Polyclonal ND 1:5000
CFP C193 Rabbit Polyclonal ND 1:10,000
CFP minus LAM C293 Rabbit Polyclonal ND 1:1000
CFP10 (Rv3874) α-CFP10 Rabbit Polyclonal ND 1:50,000
Cytosol minus LAM α-CYTO(-)LAM Rabbit Polyclonal ND 1:20,000
DnaK (Rv0350) IT-40 (HAT1) Mouse Monoclonal IgM 1:50
IT-41 (HAT3) Mouse Monoclonal IgG1 1:500
ESAT6 (Rv3875) α-ESAT6 Rabbit Polyclonal ND 1:5000
GlcB α-GlcB Mouse Monoclonal IgG1 1:50
GlnA (Rv2220) IT-58 (CBA5) Mouse Monoclonal IgG1 1:20
GroEL2 (Rv0440, 65kDa) IT-13 (TB78) Mouse Monoclonal IgG1 1:100
IT-56 (CBA1) Mouse Monoclonal IgG1 1:50
IT-70 (DCA4) Mouse Monoclonal IgG1 1:200
CS-44 Mouse Monoclonal IgG2a 1:50
GroES (Rv3418c) IT-3 (SA-12) Mouse Monoclonal IgG2a 1:20
HBHA (Rv0475) α-HBHA Mouse Monoclonal IgG2a 1:50
KatG (Rv1908c) IT-42 (HBT1) Mouse Monoclonal IgG2a 1:10
IT-57 (CDA4) Mouse Monoclonal IgG1 1:20
LAM (lipoarabinomannan from H37Rv) CS-35 Mouse Monoclonal IgG3 1:20
CS-40 Mouse Monoclonal IgG1 1:20
α-LAM Rabbit Polyclonal ND 1:1000
LAM (lipoarabinomannan from M. smegmatis) 906.4321 Mouse Monoclonal IgG3 1:20
H37Rv live infection Guinea Pig Polyclonal ND 1:1000
CSU93 live infection Guinea Pig Polyclonal ND 1:1000
MPT32 (Rv1860, 45kDa glycoprotein, ModD, Apa) CS-93 Mouse Monoclonal IgG1 1:20
α-Rv1860 Rabbit Polyclonal ND 1:5000
MPT51 (Rv3803c, FbpD) IT-52 (HBT4) Mouse Monoclonal IgG1 1:5
Pgi (Rv0946c) IT-43 (HBT3) Mouse Monoclonal IgG2a 1:20
PhoS1 (Rv0934, 38kDa, PstS1) IT-15 (TB72) Mouse Monoclonal IgG1 1:20
IT-21 (HYT28) Mouse Monoclonal IgG1 1:5
IT-23 (TB71) Mouse Monoclonal IgG2a 1:100
IT-47 (HBT12) Mouse Monoclonal IgG1 1:20
Rv1411c (P27, LprG) α-Rv1411c Mouse Monoclonal IgG1, IgG2b 1:50
Rv2780 (Ald) IT-46 (HBT10) Mouse Monoclonal IgG1 1:500
SodA (Rv3846) CS-18 Mouse Monoclonal IgG1 1:50
Whole Cell Lysate E193 Rabbit Polyclonal ND 1:2000
Whole Cell Lysate minus LAM E293 Rabbit Polyclonal ND 1:24,000
Research materials produced from mycobacterium spp. are distributed by the biodefence and emerging infectious research resources repository (BEI Resources).
*Please visit the BEI Resources Website for complete ordering instructions.

Individual SOPs: Standard Procedures