To begin, prepare Selenoprotein solution Using thermal shift assay or TSA buffer. dispense the protein, small molecules, and SYPRO Orange dye to the appropriate wells of a 384-well plate. Cover the plate with optically clear adhesive film and briefly spin down the plate at 1, 000 g for one minute in a centrifuge.
Then place the plate in a real-time PCR machine and program it to hold the sample at 20 degrees Celsius for two minutes, followed by a 0.5 degree increase in temperature per minute up to 95 degrees Celsius. Export the data from the RT PCR machine for relative fluorescence units or RFU with respect to temperature. Using the software of choice, extract and plot data points up to the highest intensity measured for each melting curve.
Determine the melting temperature with the Boltzmann sigmoidal fit for the data to determine the melting temperature or Tm.The thermal denaturation curves indicated a four degrees Celsius increase Escherichia in the thermal stability of Escherichia coli SelO in the presence of ATP with magnesium ions and a 12 degrees Celsius rise in the same for ATP with manganese ions. However, there was no shift in thermal stability upon incubation with UTP, indicating that Escherichia coli SelO may not exhibit detectable binding to UTP. The no protein as well as the no dye controls had low fluorescence signal, which was not responsive to the temperature increase.
