PROTEOSTAT^® Protein refolding and aggregation sensing kit

Figure 1. Refolding Lysozyme using a Redox Environment. The experiment was designed according the DOE matrix (in product insert, Table 1). In detail, 20 mg/mL lysozyme was first denatured in the 6M Guanidine solution at 4°C for 18 hours. Then, the denatured lysozyme was diluted 1: 20 into the various refolding buffers and incubated overnight at 4°C. For the PROTEOSTAT^® Protein Refolding and Aggregate Sensing assay, a 1:20 dilution of the refolding solutions was added directly to PROTEOSTAT^® Assay Buffer containing dye and fluorescence intensity was determined (Ex = 550 nm , Em = 610 nm). The enzymatic activity of the lysozyme in the refolding buffers was measured using 20 µg/mL Micrococcus lysodeikticus cell walls that had been excessively labeled with fluorescein, which caused dye quenching. When the lysozyme acts on this substrate, unquenched dye-labeled fragments of cell wall are released. The fluorescence intensity increase was determined (Ex/Em 490/520 nm). Native protein diluted in PROTEOSTAT^® Assay Buffer was used as a reference for both the aggregation and activity assays. The refolding solutions with high aggregation signal typically had low enzymatic activity and thus should be avoided. 1A-15A: with DTT; 1B-15B: with GSH/GSSG.