Technical Notes

2 TECHNICAL NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. reference sample (Figure 1A). Repeated freeze-thaw cycles resulted in an increase in the initial ratio and thereby a decrease in ∆ ratio, indicating an increase in unfolded protein (Table 1). As expected, the fully denatured sample showed the greatest increased initial ratio and lacked an unfolding transition, confirming denaturation. The sample containing 50% of unfolded protein showed an unfolding transition with a reduced ∆ ratio value. Interestingly, the T i s of the two partially denatured samples (freeze-thaw and 50% denatured) were very similar to the T i of the reference sample, suggesting that a fraction of the protein population is denatured, while the remaining material is still correctly folded. Results In these experiments, p38 kinase was selected as a test protein and subjected to a series of storage- related stresses. One aliquot of the protein was repeatedly frozen at -80°C and then thawed. A second aliquot was incubated for a prolonged period at temperatures above its inflection temperature (T i ) ~of 55 °C, resulting in fully unfolded protein. Fully unfolded sample generated by thermal treatment was mixed with native p38 to create a preparation that represented 50% unfolded protein. The three sample preparations were analyzed against a reference sample of freshly prepared material using the Tycho NT.6. The generated unfolding profiles showed significant differences when compared to the 30 40 50 60 70 80 90 100 0.7 0.8 0.9 0.1 1 10 100 1,000 10,000 -30 -20 -10 0 ∆ F (‰) norm Ratio 350 nm / 330 nm Temperature (°C) SB239063 concentration (nM) A B fresh freeze-thaw cycles 50 % denatured fully denatured Tycho NT.6 unfolding profiles Monolith NT.115 dose-response curves Figure 1: Effect of storage-dependent denaturation on p38 kinase thermal unfolding profile and functionality. A) The thermal unfolding profiles of p38 kinase were generated for freshly prepared protein (teal colored line), a er 6 freeze-thaw cycles (orange- colored line), a er full thermal denaturation (purple-colored line), and of a mix containing 50% fresh protein and 50% thermally denatured protein (green-colored line). B) Dose-response curves demonstrating the interaction between the small molecule inhibitor SB239063 to each of the four protein preparations described in A determined using Microscale Thermophoresis (MST). Experiments were performed on the Monolith NT.115 system.

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