Maximizing the amount of DLS data from a single thermal ramp experiment means you won’t miss anything about your protein’s behavior. The newest version of Uncle software keeps the time spent moving and adjusting lasers and detectors to a minimum, dedicating more time to gathering data. The results speak for themselves – Sizing with Thermal Ramp now has a 40% faster read rate than before. That means each read is done in about 12 seconds, then it’s time to move on the next sample.
By collecting DLS data during a thermal ramp, Uncle determines the average particle size of an IgG sample and monitors the thermal denaturation of the protein. Antibody unfolding and aggregation from heating is shown by an increasing Z-average diameter as temperature increases (Figure 2).
Uncle uses Tsize as a metric to compare increases in average particle size across multiple samples.
Tsize is the temperature at which the average particle size begins to increase. Proteins and formulations that resist unfolding and aggregation tend to have higher Tsize values. In this example, higher concentrations of arginine in the buffer protect the antibody from aggregation and therefore result in higher Tsize values.1
Uncle hunts for aggregation and reports the Tagg 660 metric to identify changes in aggregation behavior. Tagg 660 uses the intensity of the scattered light from a DLS measurement to track aggregation in the same manner as an SLS measurement. Just like with Tsize values, Tagg 660 values increase with higher concentrations of arginine (Figure 3A).
Uncle determines the values of both metrics for every sample when you use the Sizing with Thermal Ramp application, providing complementary metrics for evaluating protein stability.
Along with the Sizing with Thermal Ramp application, Uncle’s Tm & Tagg with Optional DLS application includes SLS measurements at 266 nm, giving a Tagg 266. Light scattering measurements with shorter light wavelengths are more sensitive to the formation of small particles, while longer light wavelengths can detect larger particles. Thus, Tagg 266 and 660 can identify the onset of aggregation of smaller and larger protein aggregates, respectively. Formulations with higher concentrations of arginine had higher Tagg 266 values, indicating delayed formation of small aggregates (Figure 3B). Since SLS at 266 nm detects the formation of small aggregates early in thermal denaturation, Tagg 660 was higher than Tagg 266 in all 3 formulations. Uncle’s toolbox of applications sharpen your view of your protein’s unfolding and aggregation behavior.