Cells of the ΔmetVF mutant were grown either on 20 mM of fructose or 20 mM of fructose + 80 mM of glycine betaine in 0.5–3 L of bicarbonate‐buffered complex medium to late exponential growth phase (on 20 mM of fructose, OD600 of 0.25; on 20 mM of fructose +80 mM of glycine betaine, OD600 of 1.5). After harvest by centrifugation (Avanti™J‐25 and JA‐10 Fixed‐Angle Rotor; Beckman Coulter, Brea, CA) at 8000 rpm and 4°C for 10 min, cells were washed with 30 mL of buffer containing 50 mM of imidazole (pH 7.0), 20 mM of KCl, 20 mM of MgSO4, 4 mM of DTE and 4 μM of resazurin and pelleted by centrifugation at 8500 rpm and 4°C for 10 min (Avanti™J‐25 and JA‐25.50 Fixed‐Angle Rotor; Beckman Coulter, Brea, CA). Subsequently, the cells were resuspended in 5 mL of imidazole buffer and transferred to 16‐mL Hungate tubes. All steps were performed under strictly anoxic conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI) filled with N2/H2 (96%–98%/2%–4%; v/v). The gas phase of the cell suspensions was changed to 100% N2 to remove residual H2 from the anoxic chamber. The total protein concentration of the resting cells was determined according to Schmidt et al. (1963 ).
Ja 10 fixed angle rotor
Manufactured by Beckman Coulter
Sourced in United States
The JA-10 Fixed-Angle Rotor is a laboratory centrifuge rotor designed to perform high-speed separations. It is capable of accommodating sample volumes up to 50 mL and can achieve maximum relative centrifugal forces (RCF) of up to 30,000 x g. The rotor is constructed of high-strength aluminum alloy for durability and efficient heat transfer.
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6 protocols using ja 10 fixed angle rotor
1
Anaerobic Cultivation of Δmetavf Mutant
2
Preparation of Anaerobic Resting Cells
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Preparation of resting cells were performed under anoxic condition. Cells of T. kivui wild type and ∆cooS were grown on glucose or on glucose + 100% CO in the headspace, in 500 ml of complex media to mid exponential phase and were harvested by centrifugation (AvantiTMJ-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 11,500 × g, 4 °C for 10 min. The supernatant was discarded and the cells were washed three times with imidazole buffer (50 mM imidazole, 20 mM MgSO4, 20 mM KCl, 20 mM NaCl, 4 mM DTE, 4 μM resazurin, pH 7). After centrifugation, cells were resuspended in 5 ml of same buffer and kept in 16 ml gas tight Hungate tubes. The headspace of the Hungate tubes were changed to 100% N2. The protein concentration was measured according to (Schmidt et al. 1963 (link)).
3
Anaerobic Cultivation and Harvest
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Cells were cultivated either on 60 mM fructose + 100 mM formate or 50 mM glycine betaine + 10% CO in 1 l bicarbonate-buffered complex medium to the late exponential growth phase (on 60 mM fructose + 100 mM formate, OD600 of 1.5; on 50 mM glycine betaine + 10% CO, OD600 of 0.7). Cells were harvested by centrifugation (Avanti J-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 8,000 rpm and 4 °C for 10 min, washed with 30 ml of buffer containing 50 mM imidazole (pH 7.0), 20 mM KCl, 20 mM MgSO4, 4 mM DTE and 4 µM resazurin and pelleted by centrifugation at 8,500 rpm and 4 °C for 10 min (Avanti J-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States). Subsequently, the pellets were resuspended in 5 ml imidazole buffer and transferred to 16-ml Hungate tubes. All steps were performed under strictly anoxic conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N2/H2 (96–98%/2–4%; v/v). To get rid of residual H2 from the anoxic chamber, the gas phase of the cell suspensions was changed to 100% N2. The total protein concentration of the cell suspensions was measured as described before (Schmidt et al. 1963 (link)).
4
Cultivation and Preparation of Anaerobic Cells
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Cells were cultivated on 50 mM glycine betaine in 0.5 to 2 l carbonate buffered complex medium to late exponential growth phase (OD600 of 0.3) and then harvested by centrifugation (Avanti J-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 8000 rpm and 4 °C for 10 min. The harvested cells were subsequently washed with 30 ml of buffer containing 50 mM imidazole (pH 7.0), 20 mM KCl, 20 mM MgSO4, 4 mM DTE and 4 µM resazurin by centrifugation at 8500 rpm and 4 °C for 10 min (Avanti J-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) and resuspended in 5 ml of imidazole buffer and kept in 16-ml Hungate tubes. All the steps were performed under strict anoxic conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N2/H2 (96–98%/2–4%; v/v). The total protein concentration in the resting cells was determined according to a previously described method [38 (link)].
5
Growth and Harvesting of T. kivui
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A 500 ml cultures of T. kivui were grown in complex or defined medium to late exponential growth phase (OD600 of 1.7 to 2.3) and then harvested by centrifugation (AvantiTMJ-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 7,000 × g and 4°C for 10 min. The harvested cells were washed with 30 ml of the respective medium by centrifugation at 8,500 rpm (5948 × g) and 4°C for 10 min (AvantiTMJ-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States). Then, the cells were resuspended in 5 ml of the respective medium and kept in 16 ml Hungate tubes. Resuspended cells were distributed into in Hungate tubes to a final volume of 10 mL and a final protein concentration of 10 mg ml–1. All the steps were performed under strictly oxygen free conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N2/CO2 (80/20; v/v) for carbonate medium or with 100% N2 for carbonate free medium. As substrate, 25 mM glucose or 25 mM mannitol was added to the resting cells. The experiment started with incubation at 65°C in water bath with shaking (150 rpm). 0.8 ml subsamples were taken for determination of protein, substrate and product concentration. The total protein concentration in the cell suspension was measured using the method by Schmidt et al. (1963) (link).
6
Anaerobic Growth and Harvesting of Acetobacterium woodii
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Cells of A. woodii wild type, Δfhs2, ΔfdhC and Δfhs2/fdhC were grown on 20 mM fructose in 500 ml complex medium to late exponential growth phase (OD 600 of 1.2 to 1.5) and then harvested by centrifugation (Avanti™J-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 7000 Â g and 4 C for 10 min. The harvested cells were washed twice with 30 ml of buffer containing 50 mM imidazole (pH 7.0), 20 mM KCl, 20 mM MgSO 4 , 4 mM DTE and 4 μM resazurin by centrifugation at 8500 rpm (5948 Â g) and 4 C for 10 min (Avanti™J-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States). Then, the cells were resuspended in 5 ml of buffer and kept in 16 ml Hungate tubes. All the steps were performed under strictly oxygen free conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N 2 /H 2 (96%-98%/2%-4%; v/v). After taking out of the anoxic chamber, the headspace of Hungate tubes filled with resting cells was changed to 100% N 2 . The total protein concentration in the resting cells was measured using the method by (Schmidt et al., 1963) .
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