2.5 Protein aggregation assay
Each type of recombinant refolded IL-18 was subjected to buffer exchange
by Vivaspin ultrafiltration unit (Satorius, Germany) toward PBS. To
generate IL-18 aggregates, 40 μg/mL of each type of protein was
incubated at 37 °C for 16-18 h in shaker incubator at 220 rpm. Protein
aggregation assay was performed by using ProteoStat Protein Aggregation
Assay (Enzo Lifesciences, USA). Protein was then loaded in triplicate in
96 well fluorescent microplates before adding ProteoStat detection dye.
The reaction was incubated for 15 min in the dark at room temperature.
The fluorescent signal was then determined at excitation at 485 nm and
emission at 620 nm by Varioskan™ LUX multimode microplate reader (Thermo
Scientific, USA)