Prometheus nt 48 series nanodsf grade high sensitivity capillaries

Buffer optimization for CHP3 was performed using nanoDSF. Buffer of purified CHP3 aliquots (100 µl was exchanged using PD SpinTrap G25 columns according to the manufacturer’s instructions. The effect of the MST labeling buffer was analyzed in both the presence and absence of 7% (v/v) DMSO as well as with either 10 mM MgCl₂ or 10 mM CaCl₂. After buffer exchange, the samples were diluted to a concentration of 3 mg/ml with the respective buffer. The nanoDSF measurement was performed using the Prometheus NT.48 device (NanoTemper) with an excitation intensity of 30%.
For analyzing the effect of pH, the buffer of 180 µl purified CHP3 was exchanged using Spin-Trap G25 columns to 150 mM NaCl and 20 mM HEPES at pH = 7.2. The protein sample was then diluted to 3 mg/ml with this buffer system. The experiment was done using conditions E1-E12 of the RUBIC buffer screen (Molecular Dimensions). For sample preparation, 8 µl of protein solution was mixed with 42 µl of each respective screening solution.
In both measurements, each sample was measured in triplicates using Prometheus NT.48 Series nanoDSF Grade High Sensitivity capillaries (Nanotemper) with an excitation intensity of 30%. The measurements were done over a range of 20 °C to 95 °C with a heating rate of 1 °C/min.

The nanoDSF Prometheus NT.48 was used to directly measure dynamic light scattering of OhyA, OhyA(E82A), and OhyA(Y201F). Briefly, 20 μl samples containing 50 mM potassium phosphate buffer, pH 6.0, 150 mM NaCl and 10 μM OhyA, OhyA(E82A), or OhyA(Y201F) were mixed and loaded into Prometheus NT.48 Series nanoDSF Grade High Sensitivity Capillaries (NanoTemper Technologies, Cat# PR-C006) by capillary force action. The capillaries were heated from 20 °C to 90 °C at a rate of 1 °C/min, and the UV light scattering was recorded to determine the apparent aggregation and T_agg.

NanoDSF experiments were performed to determine protein stability employing intrinsic tryptophan or tyrosine fluorescence of purified Elp456 complexes. Proteins were diluted to concentration 100 µg/ml in 20 mM HEPES pH 7.5, 50 mM NaCl, 2 mM DTT. Samples were loaded into Prometheus NT.48 Series nanoDSF Grade High Sensitivity Capillaries (NanoTemper Technologies) and measured using a Prometheus NT.48 at temperature range from 20 to 95 °C (LED/excitation power setting 10%, temperature slope 2 °C/min). Data was analyzed using NanoTemper software, n = 3.