Protein Protocols Protein Protocols

1048 Andrew
Fig. 1. Diagramatic representation of the major fragments of an IgG molecule that can be
produced by enzymatic digestion, as described in the text.
2. Materials
2.1. Digestion with Pepsin
1. Purified solution of mouse McAb (IgG or IgM) at a concentration of ≥1 mg/mL.
2. 0.2 M Sodium acetate buffer brought to pH 4.0 with glacial acetic acid.
3. 0.2 M Sodium acetate buffer brought to pH 4.5 with glacial acetic acid.
4. 0.1 mg/mL pepsin (Sigma [St. Louis, MO] number P6887) in acetate buffer at appropriate
pH (see Subheadings 3.1. and 3.2.).
5. 2 M Tris base.
6. Phosphate-buffered saline (PBS) brought to pH 8.0 using NaOH.
7. Protein A-Sepharose CL-4B (Sigma number P 3391) swollen in PBS, pH 8.0, and packed
into a 10 × 100 mm column, or a 1-mL HiTrap Protein A column (Sigma number 5-4838).
8. PBS.
9. A size exclusion column equivalent to 26 × 900 mm Sephacryl S-200 (Pharmacia, Uppsala,
Sweden).
10. Centriprep 100 concentrator (Amicon, Beverly MA; Stonehouse, Glocestershire, UK).
2.2. Digestion with Papain
1. Purified solution of mouse IgG McAb at a concentration of ≥1 mg/mL.
2. PBS made to 0.02 M with respect to ethylenediaminetetra-acetic acid (EDTA) and 0.2 M
with respect to cysteine.
3. Iodoacetamide crystals.
4. PBS brought to pH 8.0 using NaOH.
5. Protein A-Sepharose CL-4B (Sigma number P 3391) swollen in PBS, pH 8.0, and packed
into 10 × 100 mm column, or a 1 mL HiTrap Protein A column (Sigma number 5-4838).
6. PBS.
7. A size exclusion column equivalent to 26 × 900 mm Sephacryl S-200 (Pharmacia) (optional).