Protein Protocols Protein Protocols

1050 Andrew
3.3. Preparation of Fab from IgG
Again, there is a variable susceptibility to the enzyme between the IgG subclasses.
The method described is one that should give good results in most cases. If fragments
are not obtained, variations in the concentration of enzyme or time of incubation can be
tried. See Notes 1, 2, and 4 for further information.
1. Use IgG in PBS at a concentration between 1 and 5 mg/mL.
2. Dissolve sufficient papain in an equal volume of digestion buffer to the volume of antibody
solution to give a papain-to-antibody ratio of 120 (ww).
3. Add the two equal solutions, one containing antibody and one containing papain together;
mix thoroughly but gently.
4. Incubate for 4–6 h at 37°C.
5. Stop the reaction by adding crystalline iodoacetamide to make the mixture 0.03 M with
respect to iodoacetamide, and dissolve by mixing gently.
6. Dialyze the mixture against PBS, pH 8.0, overnight at 4°C.
7. Equilibrate the protein A column in PBS, pH 8.0, then add the dialyzed mixture to it. Collect
the unbound fraction. Wash the column with PBS, pH 8.0, to completely recover the Fab
fragments.
8. Concentrate the Fab fragment mixture to a volume