Preparation of Fab see Note

1. Add 1/10 vol of 1 M sodium acetate, pH 5.0, to an IgG solution of approx 5 mg/mL and check that the resulting pH is between 5.0 and 5.5. If not adjust by the careful addition of 0.1M HCl.

2. Add 1/100 vol of 1 M cysteine and a 1/50 vol of 50 mM EDTA.

3. Add 10 ^g of papain per mg of IgG and incubate at 37°C for 6-12 h (see Note 27)

4. Inactivate the papain by the addition of iodoacetamide to a final concentration of 25 mM.

5. Fab can be purified using protein A agarose (see Subheading 3.2.3.3.). Fc and any undigested antibody should bind to the column matrix. If further purification is required, anion exchange chromatography on DEAE-Agarose (Subheading 3.2.1.2.) or size-exclusion chromatography (Subheading 3.3.3.) are options.

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