Thermostatc

For untargeted GC–MS analysis, the standard Kundu et al. method was followed [26 (link)]. An extract of 480 µL of pure methanol along with 20 µL of 0.2 mg mL⁻¹ ribitol (adonitol) as internal standard was added to 20 mg of dried callus and shoot sample. The mixture was vigorously shaken for 2 min and then heated at 70 °C for 15 min (ThermoStatC, Eppendorf, Hamburg, Germany). To this, an equal volume of water was added and vortexed (Spinix vortex shaker, Tarsons, Mumbai, India), followed by the addition of 250 µL of chloroform and thoroughly mixed. This mixture was centrifuged (Eppendorf ^R centrifuge 5430 R) at 2200× g for 10 min at room temperature (~22–25 °C). The upper aqueous phase was pipetted out and dried in a speed vacuum rotator (Concentrator plus, Eppendorf) at 45 °C for 2.5 h. The dried fraction was redissolved in 40 µL of 20 mg mL⁻¹ methoxamine hydrochloride prepared in pyridine and then incubated for 90 min at 30 °C (ThermoStatC, Eppendorf). A total of 60 µL of MSTFA (N-methylN-(trimethylsilyl) trifluroacetamide) was added to the above solution and incubated for 30 min at 37 °C. A total of 100 µL of this derivatized sample was transferred in an insert containing a GC–MS glass vial and stored at 4 °C until it was analyzed in GC–MS/MS (TQ8050 NX, Shimadzu, Kyoto, Japan).

The materials used in this study were purchased from Sigma Aldrich, namely BSA with a purity of ≥98% (product No. A7906) and YCl₃ with a purity of 99.99 % (product No. 451363). SA is one of the most abundant blood proteins in mammals and has a net negative charge of −10e at neutral pH^{46 (link)}. The protein solutions were prepared with degassed Milli-Q water at c_p of 5, 20, and 50 mg/ml and T ranging from 10 to 40 °C. c_s was varied from 0 to 60 mM. While in this study we focus on BSA, we emphasize that the effects are expected to be rather general, as indicated in tests with, e.g. BLG.
The temperature-dependent phase diagram shown in Fig. 1(a) was generated with the Thermostat C of Eppendorf for a stable temperature control. The phase transitions were determined by eye based on onset of turbidity in Fig. S2. Typical aggregation sizes and its formation was part of a previous study by Soraruf et al.^{47 (link)}. For the lowest investigated protein concentration of c_p = 5 mg/ml, c^* and c^** had to be determined via UV-Vis spectroscopy measurements with the Cary 50 UV-visible spectrometer of Varian Technologies since the turbidity detection by eye was not sufficiently precise, see Fig. S2⁴⁵ . The spectrometer was also used to perform absorbance measurement to determine the protein stock solution concentration via the Lambert-Beer law.

The RPA NFO reaction used a modified reverse primer labeled with FAM. The sequence of the NFO probe was designed to include the internal presence of tetrahydrofurane, and a C3 blocking of the 3′ end. The HBV NFO probe was labeled with biotin and the control NFO probe was labeled with digoxigenin (Supplementary Figure S1).
The real-time RPA assay was performed in a 50 μL volume using the TwistAmp^® NFO kit (TwistDx). The 50 µL reaction mix included 29.5 μL rehydration buffer, 3 μL extracted DNA template, 2.1 μL FAM-labeled forward primer (10 µM), 2.1 μL reverse primer (10 µM), 0.6 µL HBV probe (10 µM), 0.6 µL control probe (10 µM), 9.8 μL dH₂O, and 2.5 μL magnesium acetate (280 mm). The reaction was performed at 39 °C (accuracy: ± 0.5 °C) in a thermostat C (Eppendorf). The resulting HBV amplicon was dual labeled with FAM and biotin and the resulting control amplicons were dual labeled with FAM and digoxigenin.
Signals were visualized on an immunochromatographic strip (Milenia Biotech, Gießen, Germany). Biotinylated HBV amplicons were captured by streptavidin. Capture of the internal control was performed using anti-digoxigenin antibodies. Amplicons and controls were detected using gold beads coated with anti-FAM antibodies. A migration control was present at the end of the strip and consisted of antibodies directed against immunoglobulins present on the gold beads.