Preparation of Fab Fragments Using Papain

Wash 0.5 ml of immobilized papain (Thermo Fisher) with 4 X 2 ml of 20 mM sodium phosphate, 20mM cysteine-HCl, 10 mM EDTA, pH 6.2 (digestion buffer), and finally suspend the gel in 1.0 ml of digestion buffer. 

Dissolve 10 mg of human IgG solution in 1.0 ml of digestion buffer and add it to the immobilized papain gel suspension. 

Mix the gel suspension in a shaker at 37°C for 4-48 hours. Maintain the gel in suspension during mixing. The optimal time for complete digestion varies depending on the IgG subclass 

After the required time of digestion, add 3.0ml 10mM Tris-HCl buffer, pH 8.0, to the gel suspension, mix, and then separate the digest solution from the gel by filtration or centrifugation at 2,000 g for 5 minutes. 

Apply the supernatant liquid to an immobilized protein A column (2ml gel Thermo Fisher) which was previously equilibrated by washing with 20ml of lOmM Tris-HCl buffer, pH 8.0. 

After the sample has entered the gel bed, wash the column with 15 ml of 10 mM Tris-HCl buffer, pH 8, while 2-ml fractions are collected. Monitor the fractions for protein by their absorbance at 280 nm. The protein eluted unretarded from the column is purified Fab. 

---