Preparation of F ab 2 Fragments Using Pepsin

Equilibrate by washing 0.25 ml of immobilized pepsin (Thermo Fisher) with 4 X 1ml of 20 mM sodium acetate, pH 4.5 (digestion buffer). Finally, suspend the gel in 1ml of digestion buffer. 

After the digestion is complete, add 3 ml of 10mM Tris-HCl, pH 8.0, to the gel suspension. Separate the gel from the antibody solution using filtration or by centrifugation. 

Apply the fragmented IgG solution to an immobilized protein A column containing 2ml of gel (Thermo Fisher) that was previously equilibrated with lOmM Tris-HCl, pH 8.0. 

Bound Fc or Fc fragments and any undigested IgG may be eluted from the column with 

After the sample has entered the gel, wash the column with 10 mM Tris-HCl, pH 8, while collecting 2-ml fractions. The fractions may be monitored for protein by measuring absorbance at 280 nm. The protein peak eluting unretarded from the column is F(ab )2. 

The only limitation to the use of this strategy is the necessity for the antibody molecule to be glycosylated. Antibodies of polyclonal origin (from antisera) are usually glycosylated and work well in this procedure, but other antibody preparations may not possess polysaccharide. In particular, some monoclonals may not be post- 

The following sections describe the most common chemical reactions used to create antibody—enzyme conjugates. 

Activation of Enzymes with NHS Ester-Maleimide Cross-linkers 

One note should be mentioned before proceeding when conjugating antibody molecules with (B-galactosidase, the antibody usually is activated with sulfo-SMCC first to take advantage of the indigenous sulfhydryl groups on the enzyme. Therefore, if (3-gal is being used, substitute the antibody for the enzyme mentioned in this protocol, 

SMCC-Activated Enzyme Containing Sulfhydryl-Reactive Maleimide Groups 

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