Issue link: https://resources.nanotempertech.com/i/1526380
1 4 A P P L I C A T I O N N O T E This is illustrated by samples 12 and 13, presented in Figure 4. The turbidity signal increases in Sample 13 at 60.6°C, which contrasts with the lack of such an increase in Sample 12 throughout the thermal ramp. In Table 2, the three highest temperatures of onset of aggregation are indicated in red which demonstrates that no large aggregates were formed below 90°C. It can thus be deduced that the protein exhibits the highest thermal stability in these three nanodiscs. Together with the data obtained from the T onset r H , it becomes evident that the copolymer AASTY 11-45 exhibits a notable advantage in the overall stability when applied to PglB. Finally, the nanoDSF data for the sample group PglC showed clear transitions in the fluorescence ratio 350 nm/350nm window along the thermal ramp. Therefore, the IPratio was identified as the second parameter in addition to the T onset r H for copolymer screening. A review of the data presented in Table 2 indicates that SMA30 (SMA200) is the most suitable copolymer for this protein. In comparison, the copolymer AASTY 11-45 nanodiscs demonstrated comparable thermal stability of the entire nanodisc, although the unfolding of the protein exhibited less stability than that observed with SMA30 (SMA200), with a difference of 9°C. Looking at further copolymers screened with Hp PglC, additional information provided by the combination of simultaneous nanoDSF, DLS and backreflection measurements can be extracted and illustrated by a comparison of samples 9, 11, and 15 (SMA BZ35, AASTY 6-45, and AASTY 6-55). If all three copolymers show a similar T onset r H , the IP ratio for sample 9 is 8°C higher than for the other two copolymers. Moreover, as seen in Figure 5, turbidity measurements also show a different turbidity profile for sample 11 (AASTY 6-45), indicating an earlier onset for the formation of large aggregates. Thus, the combined information allows for a ranking of those three copolymers with SMALP BZ35 being the most favorable and AASTY 6-45 the least favorable for stabilizing Hp PglC.