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Dobos Lab: Subcellular Fractions

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.

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