Thermo Scientific™ Immobilized Papain (Agarose Resin)

Description

• 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