Protocols / How To: Confocal Microscopy

Protocol:

After induction of heterologous T4MBP expression, bacterial cell cultures were centrifuged for 15 min at 4500 x g and 4 °C. While the supernatant was decanted, the precipitated bacteria were dissolved in 20 ml isosmotic buffer. According to the harm of heavy metals, all medium supernatants were stored and had to be picked up by professional disposal service. Starting with the dissolving the precipitate, the washing is repeated four more times. Due to the heavy metals, the supernatants obtained here had to be stored also. As the last step, precipitated bacteria were dissolved in 1 ml H20 and dried for 2 d at 60 °C. For latter MS, the reaction tube´s dry-weight before and after loading the aqueous bacteria solution had to be determined.