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.
96 well clear flat bottom
Manufactured by Greiner
Sourced in United States
The 96 Well Clear Flat Bottom is a standard laboratory equipment used for various experimental procedures. It provides a flat-bottomed well structure with a capacity of 96 individual wells. The clear material allows for visual inspection and monitoring of the contents within the wells.
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2 protocols using 96 well clear flat bottom
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TaHAL Enzyme Activity Assay at Different pH
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TaHAL Enzyme Activity Assay at Different pH
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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.
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