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Papain Antibody Digestion Protocol

This protocol details the steps for digesting 1 mg of immunoglobulin G (IgG) antibody using papain. It ensures efficient generation of Fab and Fc fragments with precise reagent concentrations and reaction conditions.

Materials

  1. Buffers and Reagents

    • 1M Sodium Acetate, pH 5.5
    • IgG Antibody (≥1 mg/mL)
    • 100 mM L-Cysteine (prepared fresh)
    • 500 mM Iodoacetamide (prepared fresh)
    • Papain Enzyme (~25 mg/mL)
    • 500 mM EDTA, pH ~7.5

  2. Other Reagents

    • Glacial Acetic Acid
    • Distilled Deionized Water (ddH₂O)

  3. Consumables

    • Microcentrifuge Tubes
    • Pipette Tips (various sizes)
    • Protein A or G Beads
    • 30 kDa Molecular Weight Cut-Off (MWCO) Filters

  4. Equipment

    • Pipettes and Sterile Pipette Tips
    • Incubator or Water Bath (37°C)
    • Refrigerator (4°C)
    • Centrifuge
    • Spectrophotometer (for A₍₂₈₀₎ measurements)
    • Magnetic Stirrer or Vortex Mixer
    • Calibrated pH Meter

Protocol

A. Activate Papain

  1. Prepare Fresh 100 mM L-Cysteine Stock Solution

    • Weigh: 6.05 mg of L-cysteine (MW 121.16 g/mol)
    • Resuspend: In 0.5 mL of ddH₂O
    • Note: Prepare fresh to ensure activity.

  2. Prepare 1M Sodium Acetate, pH 5.5

    • Weigh: 4.1 g of Sodium Acetate powder (MW 82.03 g/mol)
    • Dissolve: In 30 mL of ddH₂O
    • Adjust pH: To 5.5 with glacial acetic acid
    • Final Volume: 50 mL with ddH₂O

  3. Prepare Papain Activation Reaction (200 µL for 10 mg IgG)

    • Components:
      • 1M Sodium Acetate, pH 5.5: 20 µL
      • 100 mM L-Cysteine: 20 µL
      • Papain (~25 mg/mL): 20 µL
      • 500 mM EDTA, pH ~7.5: 0.8 µL
      • ddH₂O: 139.2 µL
    • Procedure:
      1. In a sterile microcentrifuge tube, combine all components.
      2. Mix gently to ensure homogeneity.

  4. Incubate Papain Activation

    • Conditions: 37°C for 20 minutes

B. Digestion

  1. Prepare Digestion Reaction (for 1 mg IgG at 1 mg/mL)

    • Components:
      • IgG Antibody (1 mg/mL): 1 mL
      • 1M Sodium Acetate, pH 5.5: 110 µL
      • Papain Activation Reaction: 20 µL
      • 500 mM EDTA, pH ~7.5: 4 µL
    • Procedure:
      1. In a 1.5 ml tube, combine all components.
      2. Mix gently to ensure even distribution.

  2. Incubate for Digestion

    • Conditions: 37°C for 3 to 5 hours
    • Optional: Monitor progress via SDS-PAGE at 1-hour intervals.

C. Inactivation

  1. Prepare Fresh 500 mM Iodoacetamide Stock Solution

    • Weigh: 46 mg of iodoacetamide (MW 184.03 g/mol)
    • Resuspend: In 0.5 mL of ddH₂O
    • Note: Prepare fresh to ensure reactivity.

  2. Add Iodoacetamide to Digestion Reaction

    • Volume: 120 µL of 500 mM iodoacetamide
    • Final Concentration: 50 mM
    • Procedure:
      1. Add 120 µL of iodoacetamide to the digestion mixture.
      2. Mix thoroughly by gentle pipetting or vortexing.
      3. Incubate: At room temperature for 30 minutes.

  3. Store Inactivated Digestion Reaction

    • Condition: 4°C until further use.

D. Purification

  1. Remove Fc Fragments Using Protein A or G Beads

    • Procedure:
      1. Add 20 µL of Protein A or G beads (assuming 20mg/ml –> 0.4 mg of Fc) to the inactivated digestion reaction.
      2. Incubate: At 4°C for 1 hour to bind Fc fragments.

  2. Pellet Beads and Transfer Supernatant

    • Procedure:
      1. Centrifuge at 10,000 × g for 1 minute to pellet the beads.
      2. Carefully transfer the supernatant to a new sterile microcentrifuge tube, leaving the beads behind.

  3. Filter and Concentrate Fab Fragments

    • Procedure:
      1. Use centrifugal concentrator with 30 kDa MWCO filter to remove Iodoacetamide (or use dialysis).
      2. Wash with buffer of choice (HBS or PBS).
      3. Quality Check: Measure absorbance at 280 nm (A₍₂₈₀₎) to verify removal of iodoacetamide and assess Fab concentration. Should be ~60% of the original IgG concentration.
      4. Store: Concentrated Fab fragments at 4°C.

Papain Volume Table

Antibody Amount (mg) 1% Papain (µL) 2% Papain (µL) 3% Papain (µL) 4% Papain (µL) 5% Papain (µL) 6% Papain (µL) 7% Papain (µL) 8% Papain (µL) 9% Papain (µL) 10% Papain (µL)
0.5 mg 2 µL 4 µL 6 µL 8 µL 10 µL 12 µL 14 µL 16 µL 18 µL 20 µL
1.0 mg 4 µL 8 µL 12 µL 16 µL 20 µL 24 µL 28 µL 32 µL 36 µL 40 µL
1.5 mg 6 µL 12 µL 18 µL 24 µL 30 µL 36 µL 42 µL 48 µL 54 µL 60 µL
2.0 mg 8 µL 16 µL 24 µL 32 µL 40 µL 48 µL 56 µL 64 µL 72 µL 80 µL
2.5 mg 10 µL 20 µL 30 µL 40 µL 50 µL 60 µL 70 µL 80 µL 90 µL 100 µL
3.0 mg 12 µL 24 µL 36 µL 48 µL 60 µL 72 µL 84 µL 96 µL 108 µL 120 µL
3.5 mg 14 µL 28 µL 42 µL 56 µL 70 µL 84 µL 98 µL 112 µL 126 µL 140 µL
4.0 mg 16 µL 32 µL 48 µL 64 µL 80 µL 96 µL 112 µL 128 µL 144 µL 160 µL
4.5 mg 18 µL 36 µL 54 µL 72 µL 90 µL 108 µL 126 µL 144 µL 162 µL 180 µL
5.0 mg 20 µL 40 µL 60 µL 80 µL 100 µL 120 µL 140 µL 160 µL 180 µL 200 µL