Cell pellets corresponding to OD600 5–10 were protein extracted using the alkali extraction protocol described in [18 (link)], and samples corresponding to OD600 0.5–1 were analyzed by SDS-PAGE and western blotting (WB). Monoclonal mouse anti-His primary antibody (Sigma H1029-2ML, 1:5000 dilution) and anti-mouse-HRP secondary antibody (Santa Cruz Biotechnology sc-2060, 1:10,000 to 1:20,000 dilution), or polyclonal rabbit anti-NifU ([13 (link)], 1:5000 dilution), polyclonal rabbit anti-NifS ([13 (link)], 1:200 dilution), polyclonal rabbit anti-NifM (1:4000 dilution), and anti-rabbit-HRP secondary antibody (Sigma A0545-1ML, 1:10,000 dilution) were used.
After the immunodetection of proteins, polyvinylidene fluoride (PVDF) membranes were stained with Coomassie to determine the total protein loaded and the blotting efficiency, as described in [19 (link)].
Representative expression results are shown throughout the manuscript. For every figure, at least two transformant colonies were analyzed for each strain and the results were obtained from at least two biologically independent experiments.
After the immunodetection of proteins, polyvinylidene fluoride (PVDF) membranes were stained with Coomassie to determine the total protein loaded and the blotting efficiency, as described in [19 (link)].
Representative expression results are shown throughout the manuscript. For every figure, at least two transformant colonies were analyzed for each strain and the results were obtained from at least two biologically independent experiments.
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