摘要：

This chapter presents the procedure for purification and assaying of cysteine proteinase inhibitor of chicken egg—cystatin. Egg white cystatin can be purified almost completely as a mixture of multiple forms by affinity chromatography on carboxymethyl-papain (Cm-papain) linked to Sepharose. If necessary, the multiple forms of cystatin are separated by preparative isoelectric focusing. Cystatin can be stored at –20°C in the presence of sufficient sucrose or glycerol to prevent freezing. Cystatin can be readily quantified by radial immunodiffusion. Antisera are raised in rabbits by intramuscular injection of purified cystatin emulsified with Freund's complete adjuvant, on two occasions, 2 weeks apart. The animals are bled 2 weeks after the second injection. The specificity of the antisera is checked by double immunodiffusion against whole egg white, side fractions from the purification, and pure cystatin. If necessary, contaminating antibodies are removed by adding predetermined amounts of whole egg white or fractions to the serum, and elimination of the immunoprecipitate. The concentration of antiserum required in the agarose gel for the assays depends on the potency of the antiserum, but is typically in the order of 5% (v/v).

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