Briefly, to generate antigen binding fragment monomer (Fab), the IgG was cleaved at 37 °C overnight with 1:100 w/w of 2x crystalline Papain (Sigma) in the presence of 1 mM EDTA (Sigma), 10 mM Cysteine (Sigma) in 20 mM phosphate buffer (pH 6.3). The digestion reaction was quenched by adding 75 mM of freshly prepared iodoacetamide. The undigested IgG and IgG Fc fragments were affinity removed by incubation with ProetinG agarose resin by incubation at 4 °C kept at constant shaking. Beads were washed with 1X PBS to remove excess Fab. The resultant Fab was buffer exchanged with 1X PBS (pH 7.4).
Crystalline papain
Manufactured by Merck Group
2x crystalline Papain is a laboratory product manufactured by Merck Group. It is an enzyme derived from the papaya plant that is commonly used in various research and analytical applications. The core function of 2x crystalline Papain is to catalyze the hydrolysis of peptide bonds in proteins.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using crystalline papain
1
Fab Generation from IgG Cleavage
2
Fab Monomer Generation from IgG
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Briefly, to generate antigen binding fragment monomer (Fab), the IgG was cleaved at 37 °C overnight with 1:100 w/w of 2x crystalline Papain (Sigma) in the presence of 1 mM EDTA (Sigma), 10 mM Cysteine (Sigma) in 20 mM phosphate buffer (pH 6.3). The digestion reaction was quenched by adding 75 mM of freshly prepared iodoacetamide. The undigested IgG and IgG Fc fragments were affinity removed by incubation with ProetinG agarose resin by incubation at 4 °C kept at constant shaking. Beads were washed with 1X PBS to remove excess Fab. The resultant Fab was buffer exchanged with 1X PBS (pH 7.4).
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