Infinite m200 pro microplate reader

TaHAL (0.1 μM) or fusion constructs (OsmY-TaHAL [0.1 μM], GB1-TaHAL [0.14 μM], DsbA-TaHAL [0.18 μM], or MBP-TaHAL [0.15 μM]) and HdeA (4 μM, if applicable) were incubated for 2 h in a pre-adjusted buffer A at the respective pH (between 7.2 and 2.2) before neutralization. The activity measurements for these samples were performed in 96-well plates (Corning® 96 Well Clear Flat Bottom or Greiner UV-Star® 96 well plates or Corning^® UV-Transparent Microplates) in a plate reader (Tecan Infinite M200 PRO Microplate Reader or EnSight, PerkinElmer, USA). After the addition of histidine (0.5 mM final concentration), absorbance was measured at 277 nm at intervals of 60 s for 2 h. When relevant, BSA (1 μM in buffer A) and PolyP (0.5 mM in buffer A) were added with the TaHAL solution into the buffer at different pH values. Similarly, TaHAL (0.1 μM) was incubated with different concentrations of HdeA (0.2, 2, and 4 mMμ) for 2 h in buffer A at either pH 7 or 2.8. The activities of the enzymes were determined as outlined above. Normalization was done in comparison with the activity values of the respective enzyme and additives at pH 7.