Preparation of Fab2

1. Add 1/10 vol of 1 M sodium citrate, pH 3.2, to an IgG solution of approx 5 mg/mL (see Notes 27 and 28) and check that the resulting pH is below 3.8. If not adjust by the careful addition of 1 M HCl.

2. Add 5 ^g of pepsin per mg of IgG and incubate at 37°C for 18 h (see Note 29).

3. Add a 1/10 vol of 5 M Tris-HCl to stop the reaction.

4. Purification of the (Fab')2 fragment can be achieved by the use of size exclusion chromatography (Subheading 3.3.3.).

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