Application Notes

5 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. heating rate of 1 °C/min. Measurements were performed within ~70 minutes, with a total sample consumption of 400 µl (10 µl for each buffer condition + 4 control experiments for each isoform without additive), and a total amount of protein of just 80 µg. The plots of the tryptophan fluorescence ratios clearly show that each additive increased T m of PPA and TAKA in a concentration dependent manner. For PPA, trehalose was most effective already at concentrations of 30 % (+ 12 °C), while glycerol was least effective, increasing T m by just 7.5 °C at a concentration of 40 % (Figure 5A and B). For TAKA, addition of 40 % sucrose proved to be most effective in increasing T m (+ 12 °C), while glycerol and trehalose showed the smallest effect (+ 7.5 °C and + 8°C, respectively) (Figure 6A and B). These results are in good agreement with a previous study investigating the effect of additives on Bacillus α-amylase thermal unfolding [12]. Conclusions In this case study, we demonstrate the performance of the Prometheus NT.48 in determining thermal unfolding properties of two α-amylase isoforms in screening approaches. By detecting changes in tryptophan fluorescence at two defined wavelengths, T m values of the amylase proteins could be determined under different conditions. All results show a good agreement with published values. Most notably however, when compared to other methods to measure intrinsic fluorescence, both sample consumption and the time required to perform the experiments are dramatically reduced by using the Prometheus. The capillary format of the instrument allows for a flexible experiment design, measuring any number of samples between 1 and 48 simultaneously. Importantly, Figure 3: Precision and reproducibility of Prometheus NT.48 unfolding data. (A) The plots represent an overlay of 10 independently recorded melting curves of PPA and TAKA, respectively. (B) Determination of Tm for both proteins displays a small standard deviation between experiments (≤ 0.2 °C) and a good correlation with published results [9]. 10 20 30 40 50 60 70 80 90 100 Maximal reproducibility: Temp (°C) F 350 / F 330 Average T values of a-amylase m from different species: Protein T (°C) m Literature T m value (°C) Aspergillus oryzae 71.3 +/- 0.20* 70 pig pancreas 63.6 +/- 0.17* 65 * SD from 10 capillaries 10 melting curves each from Aspergillus oryzae- and pig pancreas a-amylase A B 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 0.90 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 10 20 30 40 50 60 70 80 90 100 Maximal reproducibility: Temp (°C) F 350 / F 330 Average T values of a-amylase m from different species: Protein T (°C) m Literature T m value (°C) Aspergillus oryzae 71.3 +/- 0.20* 70 pig pancreas 63.6 +/- 0.17* 65 * SD from 10 capillaries 10 melting curves each from Aspergillus oryzae- and pig pancreas a-amylase A B 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 0.90 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 B Average T m values of α-amylase from different species A B Reproducibility testing of PPA and TAKA Temp (°C)

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