*5.7. MicroScale Thermophoresis Measurements*

Further, MST experiments were conducted to find the affinity of Mg2+ to MTase by following the manufacturer's protocol {Monolith NT.115 Series, NanoTemper Technologies). The process involved fluorescent labelling of MTase using a His-tag labelling kit (NanoTemper Technologies). Briefly, 50 nM of the enzyme was incubated with the labelling buffer at RT for 30 min. For Mg2+, the two-fold serial dilutions were made starting from 5 mM to 300 nM in 16 steps. The assay was performed in buffer (20 mM HEPES, 100 mM NaCl, 0.08% NLS, and 5% glycerol) that provides a good stability strength for protein and Mg2+. The mixture was loaded into glass capillaries and the MST analysis was performed using Monolith NT.115 (NanoTemper Technologies) at 25 ◦C.
