Immobilized porcine pepsin is ideal for digestion of IgG to generate F(ab')2 fragments. Pepsin is a non-specific endopeptidase that cleaves peptide bonds preferentially at aromatic residues such as tyrosines and phenylalanine. It generally does not cleave at alanine, glycine or valine residues.

Immobilized Pepsin Digestion Protocol
1. Make a digestion buffer consisting of 0.2M Sodium Acetate, pH 4.4 (or other suitable buffer).
2. If IgG is salt free dissolve 10 mg of your protein sample in 1ml digestion buffer, otherwise dialyze IgG against digestion buffer then concentrate to 10mg/ml.
3. Wash 0.25ml of resuspended immobilized pepsin slurry with 2x4ml of digestion buffer. Separate the gel from the buffer after each wash by centrifugation or by column. Discard buffer after each wash.
4. Resuspend the washed immobilized pepsin gel in 0.5ml of digestion buffer then add 1ml of your protein sample to the resin.
5. Incubate mixture in a shaking water bath for 4-5 hours at 37C with rapid agitation.
6. Separate the immobilized pepsin gel from the digestion mixture as noted in step 3. Retain supernatant as your pepsin-digested protein sample.
7. Maximum fragment recovery can be obtained by washing immobilized pepsin with 1-2ml of 0.01M Tris-HCl, pH 7.5. Add wash to digest recovered in step 6. Used immobilized pepsin resin should be discarded and not reused.

Note: Optimization of immobilized pepsin protocol is required for specific applications, antibody species, and subclasses. Recommended reaction conditions are pH 1 to 4.5 at 20C to 37C. The reaction rate will be increased by increasing the enzyme to protein substrate ratio and incubation temperature.