Western blotting was carried out using standard procedures as described in detail by Chen and Gottesman (2016) (link). Antibody dilutions used were polyclonal rabbit anti-MutS antibodies (1:2500; Gene Check, Inc.), monoclonal mouse anti-EF-Tu antibody (1:10,000; LifeSpan BioSciences, Inc.), polyclonal rabbit anti-RpoS antibodies (1:5000) (Battesti et al. 2015 (link)), polyclonal mouse anti-RpoD antibodies (1:5000), polyclonal rabbit anti-Hfq antibodies (1:2500) (Moon and Gottesman 2011 (link)), anti-rabbit AP-conjugated IgG (1:5000; Cell Signaling Technology), and anti-mouse AP-conjugated IgG (1:10,000; Santa Cruz Biotechnology). Western blot images were acquired by capturing the chemifluorescent signals using the Image Analyzer LAS-3000 (Fujifilm Life Science), ensuring signals in the linear range. The intensity of bands was quantified by using ImageStudio software and normalized to the signals of EF-Tu as a loading control.
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Quantitative Analysis of Protein Levels
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Corresponding Organization :
Other organizations : National Cancer Institute, Center for Cancer Research
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Variable analysis
independent variables
- Polyclonal rabbit anti-MutS antibodies (1:2500)
- Monoclonal mouse anti-EF-Tu antibody (1:10,000)
- Polyclonal rabbit anti-RpoS antibodies (1:5000)
- Polyclonal mouse anti-RpoD antibodies (1:5000)
- Polyclonal rabbit anti-Hfq antibodies (1:2500)
- Anti-rabbit AP-conjugated IgG (1:5000)
- Anti-mouse AP-conjugated IgG (1:10,000)
- Intensity of bands quantified by ImageStudio software and normalized to the signals of EF-Tu as a loading control
- Standard procedures for Western blotting as described by Chen and Gottesman (2016)
- Signals in the linear range captured using the Image Analyzer LAS-3000 (Fujifilm Life Science)
- EF-Tu as a loading control
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