Western blot assays were done essentially as described previously (51 (link)). Primary polyclonal rabbit antibodies were anti-GFP (1:15,000 dilution; Invitrogen), anti-FLAG (1:1,500 dilution; Sigma), anti-TraA (1:25,000 dilution) (11 (link)), and anti-TraB (1:15,000 dilution) (9 (link)). For chemiluminescence detection, horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody was used (1:20,000; Pierce). Blots were washed and developed using a KwikQuant imager (Kindle Biosciences LLC).
To image fluorescently tagged TraA and TraC, cells were spotted onto custom-made 0.8% agarose pads in TPM buffer and imaged using a Nikon E800 microscope with a 100× oil immersion lens. Images from GFP and mCherry filter sets were processed and overlaid using Image-Pro Plus software.
To image fluorescently tagged TraA and TraC, cells were spotted onto custom-made 0.8% agarose pads in TPM buffer and imaged using a Nikon E800 microscope with a 100× oil immersion lens. Images from GFP and mCherry filter sets were processed and overlaid using Image-Pro Plus software.
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