CelLytic™ M

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells are harvested by centrifugation (5 min, 15,000 × g, 4 °C).	- Cell pellets are resuspended in 850 μl of lysis buffer (90 mm Tris, pH 8.0, and 100 μm fluorocitrate), and bacteria are mechanically disrupted in a Fast Prep instrument at a speed of 6.0 for 30 s. Lysates are centrifuged (1 min at 15,000 × g and 4 °C).

Upstream tips
- Cells are harvested by centrifugation (5 min, 15,000 × g, 4 °C).
Protocol tips
- Cell pellets are resuspended in 850 μl of lysis buffer (90 mm Tris, pH 8.0, and 100 μm fluorocitrate), and bacteria are mechanically disrupted in a Fast Prep instrument at a speed of 6.0 for 30 s. Lysates are centrifuged (1 min at 15,000 × g and 4 °C).

Publication protocol

Bacteria were cultivated in LB-L as described before, and aliquots of the cultures (2 ml) were removed after 3, 6, 9, and 12 h of growth. Cells were harvested by centrifugation for 5 min at 15,000 × g and 4 °C, cell pellets were resuspended in 850 μl of lysis buffer (90 mm Tris, pH 8.0, and 100 μm fluorocitrate), and bacteria were mechanically disrupted in a Fast Prep instrument (Qbiogene, Heidelberg, Germany) at a speed of 6.0 for 30 s. Lysates were centrifuged for 1 min at 15,000 × g and 4 °C, and total protein concentrations of the supernatants were determined according to the method described by Bradford. Aconitase activities were assayed in cell-free supernatants as described previously. One unit of aconitase activity was defined as the amount of enzyme necessary for a ΔA240 min−1 of 0.0033. Citrate synthase activities were determined with the Citrate Synthase Assay Kit (Sigma) according to the manufacturer's recommendations, with the following modifications. Cell pellets were suspended in CelLytic M Cell Lysis Reagent, and cell-free supernatants were obtained as described above.

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Manufacturer protocol

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