If two formulations give your protein nearly the same melting temperature, how do you decide which one deserves further testing?
As I continue learning about protein stability, I’m finding that what happens after heating can be just as useful as what happens during it.
That is what caught my attention about temperature cycling on SUPR-DSF.
What does melting temperature tell us?
Intrinsic differential scanning fluorimetry, or intrinsic DSF, tracks changes in a protein’s natural fluorescence as it heats up. These changes can reveal unfolding, the loss of its folded structure.
The apparent melting temperature, Tm, describes where an unfolding transition occurs. It is useful for comparing how proteins respond to heat under matched conditions. Cohrs et al., 2025.
However, a higher Tm does not automatically mean a better formulation. In one antibody study, Tm did not correlate with long-term aggregation rates during refrigerated or room-temperature storage. Rembert et al., 2024.
So there is another question worth asking: does the protein recover when it cools down?
What temperature cycling adds
SUPR-DSF supports modulated scanning fluorimetry, or MSF, which adds repeated heating and cooling cycles.
After each heating step, the sample returns to a reference temperature. The instrument measures whether its fluorescence returns toward the starting signal. Increasing the heating temperature across successive cycles reveals when recovery begins to fail.
This provides a nonreversibility onset temperature, Tnr, alongside the unfolding information.
There is research behind asking this question. In a study of 13 antibodies, those that resisted aggregation during storage at 40°C had higher nonreversibility onset temperatures. That makes reversibility a useful additional clue when choosing candidates for further testing. Berner et al., 2021.
Why the screening format matters
A useful measurement also needs to fit the amount of protein, time, and budget available.
SUPR-DSF uses as low as 7 µL samples in plates with up to 384 wells. Small sample volumes can help conserve material, while the plate capacity provides room for multiple buffers, additives, controls, and replicates.
It also uses standard qPCR plates and seals, which can help keep routine consumable costs down. For a lab comparing many conditions, those practical details can make a broader screen easier to manage.
How I would use the extra information
Imagine two formulations with similar Tm values, but one loses fluorescence recovery earlier. That hypothetical result gives you a reason to investigate a difference that the melting temperatures alone missed.
My takeaway: use SUPR-DSF to compare both unfolding and recovery, then use those results to decide which formulations deserve the next round of testing.
These posts share my own perspective as I continue learning and are not official company statements.
AI assisted with research and drafting this article.
Top comments (0)