Papain Actigel

Papain is a nonspecific cysteine protease, which cleaves IgG at the hinge region generating three fragments of ~ 50kDa (two Fabs and one Fc fragment). Antibodies digested with Papain enzyme cannot carry out agglutination, opsonization, precipitation, or lysis, but the Fab fragments are active and would bind and neutralize the respective antigens. Coupling of Papain with the Superflow resin increases the stability of papain enzyme, facilitates controlled digestion, and avoids papain contamination in the digested products. The Fabs can be further purified on the Protein A-Superflow column to get rid of the Fc fragments, and the flow-through will have pure and active Fabs.

Product Code: 6700
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Table 1. Resin Characteristics

Bead Material Agarose
Bead Percentage 4%
Bead Size60-160 µm
Flow Rate1>25 mL/min (>850 cm/hr) @ 25oC
pH Stability22-8
Storage Temperature2-8o C
Storage Buffer 20% Ethanol
FormSlurry
Chemical Stability2Stable in all commonly used aqueous solutions and buffers.
Physical Stability2Negligible volume variation due to changes in pH or ionic strength.

1Linear flow rate = volumetric flow rate (cm3/h)/column cross-sectional area (cm2)

2Data refer to the coupled product, provided that the ligand can withstand the pH or chemical environment. Please note the following: pH stability, long term refers to the pH interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.

Instructions for Use
Papain Actigel was developed for the production of Fab fragments from IgG molecules. It is intended to be used with Sterogene Bioseparations’ Protein A media. Papain has an optimum pH range between 6 and 7 and requires 20 mM Cysteine for activation.

Buffers:
Digestion Buffer: 20 mM Phosphate, 20 mM Cysteine, 10 mM EDTA, pH 7
Enzyme Regeneration Buffer: 0.1M Phosphate, 2 mM EDTA, 10 mM Dithiothreitol, pH 6.8
Papain Actigel Storage Buffer: 0.1M Acetate, 50% Glycerol, pH 4.5
Papain Equilibration Buffer: 20mM Tris, pH 8.5
Papain Elution Buffer: 0.1M Glycine, pH 2.8 or 0.1M Citrate, pH 2.7
Neutralization Buffer: 1M Tris base
Papain Storage Buffer: 20% Ethanol

Protocol: Regenerate Papain Actigel before first use and after > 1 month of storage.
1. Wash Papain Actigel with 10 bed volumes of deionized (DI) water.
2. Add 1.5 volumes of Regeneration Buffer and mix for one hour at room temperature.
3. Wash with 10 bed volumes of DI water.
4. Store in Papain Actigel Storage Buffer or equilibrate in 1.5 volumes Digestion Buffer.
Digestion
1. For every 20 mg of IgG use 1 mL Papain Actigel (optimal IgG concentration is 5 mg/mL in Digestion Buffer + Beads).
2. Optimize digestion time between 2-18 hours; 6 hours is typical for an IgG at 5 mg/mL at 37°C.
3. Remove supernatant and adjust pH to 8.5 with 1M Tris base.
4. Load onto Protein A media and equilibrate with 20 mM Tris, pH 8.5.
5. Collect fractions and assay for Fab – 3 bed volumes should be sufficient.
6. Store Fabs under conditions optimized for the specific Fab.
7. Elute bound material from Protein A resin with Protein A Elution Buffer until the absorbance reaches baseline.
8. Re-equilibrate Protein A medium in Equilibration Buffer until pH of eluent is 8-8.5.
9. Store Papain Actigel and Protein A medium in the appropriate storage buffer above.

To Download Instructions for use:
INST 6700 Papain Actigel

Safety Data Sheets

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