Immobilized Papain (Agarose Resin)

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 als be used to generate F(ab')2 fragments. To prepare F(ab')2 fragments, the papain is first activated with 10mM 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.

Highlights:

• Support: Crosslinked 6% beaded agarose
• Specific Activity: 16-40 BAEE units/mL papain before loading
• Loading: 250μg papain/mL gel
• Form: Slurry in 50% glycerol, 0.1M Na-acetate, pH 4.4, 0.05% sodium azide

Additional Features:

• Alternate name: Papaya peptidase I
• Cleaves somewhat nonspecifically at exposed residues
• Source: Carica papaya latex Molecular weight: 23,000
• Protease type: Cysteine protease
• For antibody fragmentation, cleaves into Fab or F(ab')2 fragments
• Reaction conditions pH 6 to 7, 20 to 37°C
• Inhibitors: heavy metals, carbonyls, NEM, p-chloromercuro-benzoate, E-64