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Cytosol
Reagent:
Cytosol Fraction, CYT
Default Quantity:
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.
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