Thermo Scientific Pierce Immobilized Ficin consists of a sulfhydryl-specific protease (ficin) that has been immobilized onto beaded agarose resin to enable controlled antibody fragmentation, especially of mouse IgG1.
Ficin, which is similar to papain, is a nonspecific, sulfhydryl protease isolated from fig latex. The molecular weight of Ficin is approximately 25,000 and it is effective at pH 4-9.5 with an optimum pH of 6.5. When IgG molecules are incubated with ficin in the presence of cysteine, one or more peptide bonds in the hinge region are split, producing either Fab fragments or F(ab´)2 fragments. Mouse IgG1 is notably difficult to digest into functional Fab or F(ab')2 fragments using papain or pepsin, however, yields and immunoreactivity are better when digesting mouse IgG1 with ficin. Immobilized Ficin was specifically designed for cleavage of mouse IgG1 into or Fab or F(ab')2 fragments, and is advantageous because it virtually eliminates autolysis, eliminates contamination of a sample with the protease and allows control of the digestion by removing the ficin or controlling the flow of sample over an immobilized ficin column.
- Can generate both Fab and F(ab')2 fragments from mouse IgG1
- Reaction can be easily controlled
- Antibody fragments are free of autodigestion products
- Ficin contamination into antibody fragments is eliminated
Immobilized ficin is more stable against heat-induced denaturation than the free enzyme, resulting in longer maintenance of activity. The Fab and F(ab')2 Preparation Kit contains immobilized ficin, other necessary reagents and an optimized protocol for mouse IgG1 digestion. Ficin will generate mouse IgG1 F(ab')2 and Fab fragments in the presence of 1 mM cysteine and 10 mM cysteine, respectively. Fragment generation from other species and isotypes is also possible through modification of the cysteine concentration and other digestion parameters.
|Properties of Ficin Protease.
||Cleaves somewhat nonspecifically at exposed residues but favors uncharged or aromatic residues
||Antibody fragmentation, cleaves into Fab or F(ab’)2 fragments
||pH 6.5, 20-37°C
||Store at 4°C
||heavy metals, carbonyls, NEM, p-chloromercuro-benzoate, E-64
- Leiner, I.E. and Friedenson, B. (1970) Ficin. Methods Enzymol. 19, 261-273.
- Kurkela, R., et al. (1988). Preparation of F(ab')2 fragments from mouse
monoclonal IgG1 suitable for use in radioimaging. J. Immunol. 110, 229-236.
- Mariani, M., et al. (1991) A new enzymatic method to obtain high-yield F(ab´)2 suitable for clinical use from mouse IgG1. Mol. Immunol. 28, 69-77.
IgG1 Fab and F(ab’)2 Preparation Kit with Immobilized Ficin
All proteases and protein-cleaving reagents (including mass spectrometry-grade)
Mass spectrometry reagents and kits for proteomics