Thermo Scientific Pierce Immobilized Papain consists of a cysteine-endopeptidase (papain) that has been immobilized onto beaded agarose resin to enable generation and purification of Fab fragments from antibodies.
Papain is a nonspecific, cysteine-endopeptidase isolated from papaya latex that is used in a wide variety of applications. When IgG molecules are incubated with papain in the presence of cysteine, one or more peptide bonds in the hinge region are split, producing three fragments of similar size: two Fab fragments and one Fc fragment. The Fc fragments can be separated from the Fab fragments using immobilized Protein A or by ion exchange chromatography.
The 50 kilodalton Fc fragment is often used for determining specificity of the Fc receptors without interference from antigen binding. Fab fragments are useful in immunohistochemical studies because the fragments penetrate the tissue better than intact IgG. Also, nonspecific binding from Fc receptors is avoided by using only the Fab portion of the immunoglobulin.
Papain can also can be used to generate F(ab')2 fragments. To prepare F(ab')2 fragments, the papain is first activated with 10 mM cysteine, which is then removed via gel filtration. If no cysteine is present during the papain digestion, F(ab')2 fragments can be generated. These fragments are often inconsistent and reproducibility is a problem.
Immobilized papain can be substituted for free papain in almost any application, 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 papain or controlling the flow of sample over an immobilized papain column. Immobilized papain is also more stable against heat-induced denaturation, resulting in longer maintenance of activity. The ImmunoPure Fab Preparation Kit contains immobilized papain, other necessary reagents and an optimized protocol for human, rabbit and mouse antibody digestion.
|Properties of Papain Protease.
||Papaya peptidase I
||Cleaves somewhat nonspecifically at exposed residues
||Carica papaya latex
||Antibody fragmentation, cleaves into Fab or F(ab’)2 fragments
||pH 6-7, 20-37°C
||Store at 4°C
||heavy metals, carbonyls, NEM, p-chloromercuro-benzoate, E-64
- Rousseaux, J., et al. (1983) Optimal conditions for the preparation of Fab and F(ab') fragments from monoclonal IgG of different rat IgG subclasses. J. Immunol. Meth. 64, 141-6.
- Coulter, A. and Harris, R. (1983) Simplified preparation of rabbit Fab fragments. J. Immunol. Meth. 59, 199-203.
- Mage, M.G. (1980) Preparation of Fab fragments from IgGs of different animal species. Meth. Enzymol. 70, 142-50.
- Rousseaux, J., et al. (1980) The differential enzyme sensitivity of rat immunoglobulin G subclasses to papain and pepsin. Molec. Immunol. 17, 469-82.
Fab Preparation Kit with Immobilized Papain
All proteases and protein-cleaving reagents (including mass spectrometry-grade)
Mass spectrometry reagents and kits for proteomics