Interaction of Protein with Promoter Fragment by MST in Solution
The interaction of CaNAC2c protein with the promoter fragment of aCaHSFA5 was analyzed by microscale thermophoresis (MST) in solution (Zillner et al., 2012). GFP in the fused protein TF-GFP was used as fluorescent label, and a CATGTG motif containing fragment within the promoter of CaHSFA5 , which was amplified by PCR with a specific primer pair and was further purified. The fragment containing the mutant version of CATGTG motif (GGGGGG) was amplified by PCR by conventional overlapping PCR-based site-directed mutagenesis. The two DNA fragments were used as the nonfluorescent molecules. The protein-DNA interactions were measured following the method of our previous study (Qiu et al., 2018). The Nano Temper Analysis 1.2.20 software was used to fit the data and determine the apparent Kd values (Papageorgiou et al., 2016; Zillner et al., 2012).