Varian carry eclipse spectrofluorometer

Fluorescence of all proteins (0.05–0.15 mg/mL) was measured at 25 °C in buffer F containing 20 mM HEPES/NaOH pH 7.6, 100 mM NaCl, 0.5 mM EDTA, and 2 mM DTT. Fluorescence was excited at 295 nm (slit width 5 nm) and recorded in the range of 310–400 nm (slit width 5 nm).
Heat-induced aggregation was monitored by heating of protein samples (0.3 mg/mL) in buffer F with a constant rate of 1 °C/min from 25 to 90 °C. The samples were illuminated at 340 nm (slit width 2.5 nm) and signal was recorded at 340 nm (slit with 2.5 nm). All fluorescent and static light scattering experiments were performed on a Varian Carry Eclipse spectrofluorometer (Agilent, Santa Clara, CA, USA).
Dynamic light scattering was performed on Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). All protein solutions were prepared in buffer F filtered through 0.2 µm filter. Experiments were performed at 25 °C at protein concentration equal to 0.3 mg/mL for HspB1, HspB5, and HspB4 and at concentration equal to 0.6 mg/mL for HspB6 and HspB8. Each experiment lasted for 10 s and was repeated 15 times. This cycle was repeated 10 times, thus providing the accumulation of 150 measurements. Number distribution was used for calculation of hydrodynamic diameter with Zetasizer software.

7.5 g of gelatin powder was dissolved into 30 mL of 80 °C Milli-Q® water by stirring. Aqueous stock solutions of either ErB or RoseB were mixed with the liquidised gelatin to reach 500 µM PS concentration before the water evaporated. The air-dried gels of 3 mm thickness were irradiated face-on with ~10⁹ protons × cm⁻² × sec⁻¹, and the overall photonic emission was registered by the iHR 320/Synapse CCD. Following light excitation (λ_ex = 530 nm for RoseB and 470 nm for ErB), the fluorescence and phosphorescence emission spectra were registered with the Varian Carry Eclipse spectrofluorometer (Agilent, Santa Clara, CA, USA), using a 5 ms gating time and 0.2 ms delay in the phosphorescence mode.