Thermal Stability Analysis of SAMHD1

Thermal melt measurements were made using the GloMelt Thermal Shift protein stability kit from Biotium on a Qiagen Rotor-gene Q qPCR instrument. Solutions of 50 mM HEPES—pH 7.5, 50 mM KCl, 5 mM MgCl₂, 0.5 mM TCEP, 3 μM SAMHD1, 1X GloMelt dye and 0/2 mM dGTPαS were subjected to a temperature gradient from 25 to 85°C at 0.5°C intervals. Each step in the gradient was held for three seconds and the fluorescence of the GloMelt dye was measured Rotor-gene Q green channel. Automatic gain optimization was performed all of the samples before execution of the temperature gradient with a signal limit of seven. Three replicates were recorded for each measured condition. Melt temperatures for each replicate were calculated using the program TSA-CRAFT (Supplemental Table S2) (29 (link)). To display differences in melt temperature between different SAMHD1 constructs, averaged traces were differentiated and normalized in Prism using 6^th order polynomial smoothing considering five neighbors on each side.