Cystatin A (CSTA) is a member of family 1 of the cystatin superfamily. Like Cystatin B, it is an intracellular inhibitor regulating the activities of cysteine proteases of the papain family such as cathepsins B, H and L. For example, immunohistochemical analysis of Cystatin A and cathepsin L is a useful indicator for malignancy in human epidermal keratinocytes. The ratio of cathepsin B and Cystatin A can be used in the differential diagnosis and treatment of patients with prostate carcinoma. The activity of recombinant mouse Cystatin A was measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC in the assay buffer 50 mM Tris, pH 7.0. Papain was diluted to 500 ug/ml in activation buffer 50 mM Tris, 5 mM DTT, pH 7.0 and incubated at room temperature for 15 minutes. The activated papain was diluted to 100 ug/ml in the assay buffer and 20 ul different concentrations of recombinant mouse Cystatin A (MW: 40.9 KD) was incubated with 20 ul 100 ug/ml papain at 37 ℃ for 10?minutes. Loading 50?μL of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50?μL of 200?μM substrate. Include a substrate blank containing 50?μL of assay buffer and 50?μL of 200?μM substrate. Then read at excitiation and emission wavelengths of 380 nm and 460 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant mouse Cystatin A significantly decreased papain activity. The inhibition IC50 was <130 nM.