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. 2021 Oct 4;12:750200. doi: 10.3389/fmicb.2021.750200

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Copyright © 2021 Carranza, Menguiano, Valenzuela-Gómez, García-Cazorla, Cabezón and Arechaga.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Immunodetection and subcellular localization of conjugative ATPases. MG1655 Escherichia coli cells were transformed with R388 wild type plasmid or with R388 encoding for TrwBmKate2, TrwCmKate2, or TrwKmKate2. Cells were grown to stationary phase and harvested by centrifugation. Cell pellets were re-suspended and lysed by mechanical methods. Soluble and membrane fractions were separated by ultra-centrifugation. Aliquots of each fraction were run in a SDS-PAGE. Gels were transferred to a nitrocellulose membrane and incubated with antibodies anti-TrwB (upper panel), anti-TrwC (middle panel), or anti-TrwK (bottom panel) and then revealed with IRDye. Upper panel: (a) wild type R388 soluble fraction, (b) R388_TrwBmKate2 soluble fraction, (c) wild type R388 membrane fraction, (d) R388_TrwBmKate2 membrane fraction, and (e) purified TrwBΔN70 protein. Middle panel: (a) wild type R388 soluble fraction, (b) R388_TrwCmKate2 soluble fraction, (c) wild type R388 membrane fraction, (d) R388_TrwCmKate2 membrane fraction, and (e) purified TrwC protein. Bottom panel: (a) wild type R388 soluble fraction, (b) R388_TrwKmKate2 soluble fraction, (c) wild type R388 membrane fraction, (d) R388_TrwKmKate2 membrane fraction, and (e) purified TrwK protein. Estimated MW values for TrwB, TrwBmKate2, and TrwBΔN70 proteins are 56.3, 82.4, and 48.6 kDa, respectively. Estimated MW values for TrwC and TrwCmKate2 proteins are 107.4 and 133.5 kDa, respectively. Estimated MW values for TrwK and TrwKmKate2 proteins are 93.8 and 119.9 kDa, respectively.