Issue link: https://resources.nanotempertech.com/i/1050485
3 APPLICATION NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Figure 2: Analysis of TAKA and PPA melting curves. (A) Plot of the tryptophan fluorescence decay upon thermal unfolding of TAKA (le ). The transition from the folded to the unfolded state is already visible in the raw fluorescence data at emission wavelengths of 330 and 350 nm. The inset demonstrates the high data point density of the Prometheus NT.48. To determine T m , the experimental data can be fitted with a polynomial function. Its first derivative displays a peak at the point of maximal slope, which corresponds to T m (right). (B) Equivalent analysis of T m for PPA. Note that in contrast to TAKA, the transition of folded to unfolded protein is not visible in the raw fluorescence data (le ), while T m can be easily determined from the fluorescence ratio plots (right). 10 20 30 40 50 60 70 80 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 10 20 30 40 50 60 70 80 90 0 500 1000 1500 2000 2500 3000 3500 20 30 40 50 60 70 80 90 0.90 0.95 1.00 1.05 1.10 1.15 10 20 30 40 50 60 70 80 90 330 nm 350 nm Fluorescence (A.U) Temp (°C) 90 330 nm 350 nm Fluorescence (A.U) Temp (°C) T m T m Temp (°C) Temp (°C) F350 nm / F330 nm Pig pancreatic a-amylase PPA („blueshift") Aspergillus oryzae „redshift") a-amylase TAKA ( First derivative analysis First derivative analysis 64 65 66 Data density B A F350 nm / F330 nm 1.20 1.25 1.30 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 Aspergillus oryzae α-amylase TAKA („redshi " ) First derivative analysis A Temp (°C) Temp (°C) 10 20 30 40 50 60 70 80 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 10 20 30 40 50 60 70 80 90 0 500 1000 1500 2000 2500 3000 3500 20 30 40 50 60 70 80 90 0.90 0.95 1.00 1.05 1.10 1.15 10 20 30 40 50 60 70 80 90 330 nm 350 nm Fluorescence (A.U) Temp (°C) 90 330 nm 350 nm Fluorescence (A.U) Temp (°C) T m T m Temp (°C) Temp (°C) F350 nm / F330 nm Pig pancreatic a-amylase PPA („blueshift") Aspergillus oryzae „redshift") a-amylase TAKA ( First derivative analysis First derivative analysis 64 65 66 Data density B A F350 nm / F330 nm 1.20 1.25 1.30 1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 First derivative analysis Pig pancreatic α-amylase PPA („blueshi " ) B Temp (°C) Temp (°C)