Antibody Fab and F(ab)₂ Fragmentation of Mouse IgG1 Kits are designed for the generation and isolation of Fab and F(ab)₂ fragments from mouse IgG₁ molecules. The kit utilises Immobilised Ficin resin. Ficin (or Ficain) (~25000 Da) is a cysteine protease enzyme (EC 188.8.131.52) isolated from fig latex that has the endopeptidase activity to cleave immunoglobulin G molecules in the hinge region. Ficin has an effective range of pH 4 to 9,5 with an optimal pH of 6,5 and cleaves bonds that involve uncharged or aromatic amino acids. Ficin is typically used to cleave mouse IgG₁ as this is difficult to cleave with papain and pepsin. In the presence of 1 to 4 mM or 10 to 20 mM cysteine, ficin generates F(ab)₂ and Fab fragments respectively.
Immobilised Ficin is a convenient reagent for producing Fab and F(ab)₂ fragments as it avoids the need to remove the ficin enzyme after digestion. Following ficin digestion the fragments are separated from undigested IgG and the large Fc fragments with the supplied Protein A Spin Column. The Protein A Resin binds the IgG and Fc molecules and the Fab or F(ab)₂ are rapidly collected due to the spin-format design. In addition, SpinOUT™ GT-600 desalting columns are supplied to ensure the initial antibody sample is in the optimal condition for fragmentation. The Fab and F(ab)₂ Fragmentation of Mouse IgG₁ Kit is optimised for mouse IgG₁, using 0,25 to 4mg/ 0,5ml sampleor using 25 to 250 μg/125 μl sample with the Fab and F(ab)₂ Fragmentation (Micro) Kit.