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Detection of CoxD in subcellular fractions of O. carboxidovorans cultivated under different conditions.

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posted on 2013-02-19, 21:43 authored by Tobias Maisel, Stephanie Joseph, Thorsten Mielke, Jörg Bürger, Stephan Schwarzinger, Ortwin Meyer

O. carboxidovorans OM5 (lanes A and B) and the insertional mutants coxD (lane D), coxE (lane E), and coxF (lane F) were grown under different metabolic conditions: lane A, chemolithoautotrophically with CO (45 CO, 5 CO2, 50 air; all values in % v/v); all other lanes, chemolithoautotrophically with H2 in the presence of CO (30 H2, 5 CO2, 30 CO, 35 air) to induce the transcription of cox-genes. In order to separate cytoplasmic fractions (S, supernatant) and membrane fractions (P, pellet), crude extracts prepared from the bacteria were subjected to ultracentrifugation (100,000× g, 2 h, and 4°C). The distribution of CoxD in the supernatant or the pellet was analyzed by applying 50 µg of protein from S or P to SDS-PAGE. Prior to PAGE all samples were boiled in 1% SDS as described in the methods section. CoxD was identified by Western blotting employing IgG antibodies raised against recombinant CoxD. The grey bands apparent from lanes A, B, E, and F originate from the CoxD protein.