pASG-IBA5

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

Protocol tips

For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

Publication protocol

For anaerobic cultures, the 400 mL of LB or M9ZB media were prepared in a 500 mL bottle that was closed with a rubber stopper, degassed with N2 and sterilized by autoclaving. The overnight aerobic culture was transferred to the medium through a syringe, without opening the bottle. In the case when the M9ZB medium was tested, cells were grown at 37 °C with shaking, and induced with AHT (0.2 μg/mL final) when the culture reached an OD600 of 0.8. When cells grew anaerobically in LB medium, they were induced at an OD600 of 0.5. The culture was supplemented with Na2MoO4 (10 mM final concentration) 15 min before induction with AHT (0.2 μg/mL final). The culture was grown for 2 h at 37 °C and then cells were pelleted. The pellet was stored at −80 °C for a few days before purification.

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Manufacturer protocol

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