RbpA was conjugated with the sulfhydryl- reactive dye, DyLight 633 Maleimide (Thermo Scientific), as described (24 (link)). Labeled RbpA (1.6 μM) was incubated with different concentrations of the σB subunit (0.8, 1.6 and 3.2 μM) in 10 μl of TB at 37°C for 10 min. Samples were analyzed on 5–10% native PAGE in Tris–glycine buffer. Gels were scanned with a Typhoon 9400 Imager (GE Healthcare).
Protocol detail
Fluorescent Labeling of RbpA
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Corresponding Organization : Centre National de la Recherche Scientifique
Other organizations : Wuhan Institute of Virology, Chinese Academy of Sciences
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Variable analysis
independent variables
- Concentration of the σ^B subunit (0.8, 1.6 and 3.2 μM)
- Binding of the labeled RbpA (1.6 μM) to the σ^B subunit
- Reaction volume (10 μl)
- Reaction buffer (TB)
- Reaction temperature (37°C)
- Reaction time (10 min)
- Native PAGE (5–10%) in Tris–glycine buffer
- Imaging method (Typhoon 9400 Imager)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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