Optical quality sealing tape

The thermal denaturation of the mutants by Differential Scanning Fluorimetry (DSF—Thermofluor) was also investigated. Solutions composed of 7.5 μl of 300 x Sypro Orange (Molecular Probes, Life Technologies, Carlsbad, California, USA) and 17.5 μl of proteins (hNGFs or hproNGFs) at the concentration of 0.7 mg/ml in Sodium Phospate 50 mM pH 7, were added to the wells of a 96-well thin-wall PCR plate (Bio-Rad, Hercules, California, USA). Buffer and Water was added as blank and control sample, respectively. The plates were sealed with Optical-Quality Sealing Tape (Bio-Rad, Hercules, California, USA) and heated in an iCycler iQ Real Time PCR Detection System (Bio-Rad, Hercules, California, USA) from 20 to 90° C in increments of 0.2°C/20 s. Fluorescence changes in the wells of the plate were monitored simultaneously with a charge-coupled device (CCD) camera. The wavelengths for excitation and emission were 485 and 535 nm, respectively. The experiment was repeated three times for each protein.
In order to compare the transition curves for the set of different proteins, each experimental set of data was normalized to the transition amplitude that is the difference between the fluorescence intensity at the start and at the maximum of the unfolding transition, respectively [28 (link)].
Data were evaluated by pairwise Student’s T-tests.

Differential scanning fluorometry was used. Aliquots (45 µL) of enzyme solution (5 µM) in 0.10 M or 1.0 M potassium phosphate buffer (pH 7.0) were mixed with 5 µL of 200 × SYPRO orange solution and aliquoted in triplicate into a 96-well-PCR plate (Bio-Rad, Hercules, CA, USA). The plates were sealed with optical-quality sealing tape (Bio-Rad) and heated in a Bio-Rad CFX Connect Real-Time PCR Detection System, from 25 °C to 99 °C in increments of 0.5 °C and hold 30 sec at each step. The excitation and emission wavelengths were 490 and 575 nm, respectively, and the fluorescence changes in the wells were monitored continuously. CFX Maestro (Bio-Rad; Version 4.0.2325.0418) was used for data processing to calculate the melting temperature T_m. Buffer without enzyme was the control.

The melting temperatures of the MhGgH variants were determined using a thermal shift (Thermofluor) assay. Each protein sample (0.5 mg ml⁻¹ final concentration) was centrifuged at 13 000g and 4°C for 15 min, mixed with 5× SYPRO Orange (Life Technologies) in storage buffer and loaded into white 96-well PCR plates (Bio-Rad) sealed with Optical Quality Sealing Tape (Bio-Rad). The plate was heated from 25 to 95°C in 0.5°C steps with 30 s hold time per step on an iCycler iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad) and the fluorescence was followed using a Cy3 dye filter (545 nm excitation/585 nm emission). Each experiment was performed in triplicate. The melting curves were analysed using the CFX Manager software (Bio-Rad) and the melting temperature was determined as the inflection point of the melting curve.

Thermal stability to unfolding of CaMan5_18 was analyzed using ThermoFluor screening [43 (link)]. The effect of pH on the melting temperature, T_m, was investigated. Reaction mixtures of 100 μL contained 2 μL of 10 mg mL^-1CaMan5_18, 2 μL SYPRO Orange dye (25x stock solution diluted 1:200 with water), and 97 μL 50 mM buffer (sodium acetate pH 5.0–5.5, sodium phosphate pH 6.0–7.5, and Tris-HCl pH 8.5–9.0), and were added to the wells of a 96-well thin-wall PCR plate (Bio-Rad). The pH value was varied from 5 to 9 in intervals of 0.5 pH units. The plates were sealed with Optical-Quality Sealing Tape (Bio-Rad), and heated in an iCycler iQ Real Time PCR Detection System (Bio-Rad) from 20–90°C in steps of 0.2°C. The changes in fluorescence were monitored with a charge-coupled device (CCD) camera. The wavelengths for excitation and emission were 490 nm and 575 nm, respectively.

To investigate the effect of pH on thermal stability for GtCel12A, 10 µM of protein in different buffer solutions were mixed with 1 µL of Sypro Orange (Invitrogen, Carlsbad, USA), used as the reporter dye, with a final concentration of 1/2000. All buffers used in the analysis were prepared at 50 mM. All measurements were performed in triplicate in a final volume of 20 µL. In the reaction control, the volume of enzyme was replaced with 19 µL of the same buffer. Mixtures were added to a 96-well thin wall PCR plate (Bio-Rad, Hercules, USA) and sealed with Optical-quality sealing tape (Bio-Rad, Hercules, USA) and incubated in an iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, USA). The temperature range tested was from 25 to 90°C, with stepwise increments of 1°C per minute and 10 s hold step for every point, followed by the fluorescence reading with excitation/emission wavelengths at 490/530 nm. Melting curve analysis and the melting temperature (T_m) determination were carried out using GraphPad Prism software (version 5.0) (GraphPad Software, La Jolla, USA).

A thermal stability assay was employed to determine the melting point (T_m) of LPLA₂ (26 (link)). An incubation mixture consisting of 2.5 μl of 8x SYPRO Orange, 1 μg of LPLA₂ in 50 mM Na citrate at pH 4.5, and ddH₂O in a final volume of 20 μl was added to wells of a 48-well thin-wall PCR plate. The plates were sealed with Optical-Quality Sealing Tape (Bio-Rad) and heated in a Real-Time PCR Detection System Life Technology (Thermo Fisher, Ann Arbor, MI) from 20 to 90°C in steps of 0.2°C. T_m values were calculated as the inflection point of the melting curve using the instrument software.

A 5000× stock solution of Sypro orange dye (Life Technologies) was diluted 50-fold in water. Solutions of 10 μL of protein buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl), 8 μL protein (2.0 mg mL⁻¹), and 2 μL of the diluted dye were placed in a 96-well PCR plate (Bio-Rad). The plate was then sealed with optical-quality sealing tape (Bio-Rad) and subjected to thermal treatment in a real-time PCR detection system (CFX96, Bio-Rad). The instrument was programed to equilibrate at 20 °C for 2 min, then increase the temperature by 0.5 °C per 30 s from 20 °C to 90 °C, and finally maintain the temperature at 90 °C for 2 min.