The metal-binding specificity of PilB was tested using ThermoFluor, a fluorescent-based method measuring changes in thermal denaturation temperature (27 (link)). Assays were done in a 96-well plate (Applied Biosystems) format. In the wells, we added to a final volume of 40 µL 1) 0 to 1 mM range of concentrations of MgCl2, MnCl2, and CaCl2, 2) 20 µM purified PilB or PilBD319A, and 3) 1/5,000 dilution of SYBR Orange (Thermo Fisher Scientific). Plates were then analyzed using a temperature gradient, from 25 to 99 °C, on a StepOnePlus RT-qPCR machine (Applied Biosystems). The data were exported in MATLAB and analyzed in GraphPad. Analyses were performed with Prism (GraphPad Software). Kd were calculated using nonlinear regression fits, applying saturation binding equation (One site − Total and nonspecific binding) using Ca2+ as nonspecific binding control.
Sybr orange
Manufactured by Thermo Fisher Scientific
SYBR Orange is a fluorescent dye used in molecular biology applications. It is designed to bind to double-stranded DNA and emit a fluorescent signal, allowing for the detection and quantification of DNA in various experiments.
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2 protocols using sybr orange
1
Determining PilB Metal Binding Specificity
2
Thermal Denaturation Analysis of PilB Binding
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The metal binding specificity of PilB was tested using ThermoFluor, a fluorescentbased method measuring changes in thermal denaturation temperature 27 . Assays were done in a 96-well plate (Applied Biosystems) format. In the wells, we added to a final volume of 40 µl (i) 0-1 mM range of concentrations of MgCl2, MnCl2 and CaCl2, (ii) 20 µM purified PilB or PilBD319A, and (iii) 1/5,000 dilution of SYBR Orange (Thermo Fisher Scientific). Plates were then analysed using a temperature gradient, from 25 to 99 ºC, on a StepOnePlus real-time qPCR machine (Applied Biosystems). The data were exported MATLAB and analysed in GraphPad. Analyses were performed with Prism (GraphPad Software). Kd were calculated using non-linear regression fits, applying saturation binding equation (One site -Total and non-specific binding) using
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