IgG Fab and Fab2 Purification

Purified IgG (1 mg) was digested to Fab and Fc fragments by exposure to 40 μg activated papain (Sigma-Aldrich) in a solution containing 10 mM cysteine, 100 mM sodium acetate pH 5.5, 125 μM EDTA for 2–4 h at 37 °C. Fab2 was produced by digestion with pepsin (Sigma-Aldrich) for 2–4 h at 37 °C. Fab and Fab2 fragments were subsequently separated from Fc by affinity chromatography using a protein A Hi-trap column (GE Healthcare) and purified to homogeneity by size exclusion chromatography (SEC)37 (link). SDS–polyacrylamide gel electrophoresis was performed to confirm the sizes of Fab and Fab2. The NativePAGE Novex Bis-Tris gel, SimplyBlue SafeStain staining solution and protein standard were purchased from Invitrogen, Inc.