Monitoring Thermally Induced Aggregation
The aggregation process can be accelerated by subjecting proteins to elevated temperature. Sheep IgG (4 mg/mL) in 100 mM sodium phosphate, pH 7.2, was maintained at 60oC with constant stirring of 400 rpm for various periods of time.
At each time point, 25 µL of IgG solution was retained for measurement. Sample was first diluted fourfold in 1x assay buffer to generate a final protein concentration of 1 mg/mL. 98 µL of test protein as well as 2 μL of the prepared ProteoStat Detection Reagent Loading Solution was added into each well. Each time point was performed in duplicate.
At the same time, the ProteoStat Protein Aggregation standards, which contain stabilized, high-quality reference samples, were used to generate a standard curve.
Figure 1 highlights thermally induced aggregation of sheep IgG, as determined using the ProteoStat dye and the Synergy Mx Multi-Mode Microplate reader. The generated curve suggests different stages associated with the formation of IgG protein aggregates.
First, a lag phase is observed during which minimal aggregation occurs. Then, a nucleation phase is observed, and aggregates start to form (growth phase). Eventually, the growth rate decreases and becomes zero, indicating exhaustion of the monomeric protein supply.