Protease activity was quantified using an azocasein assay as previously described [43, 44 (link)]. Briefly, 400 µl of filtered supernatant was mixed with 400 µl of azocasein substrate (Sigma, 5 mg ml−1 in 0.1 M pH 8 Tris-HCL buffer) and incubated for 1 h in a shaking water bath (100 r.p.m., 37 °C). After 1 h the undegraded azocasein was precipitated by adding 100 µl of trichloroacetic acid (10%) and the mixture was spun down. Subsequently 100 µl of supernatant was added to 100 µl of NaOH (625 mM) in a 96-well plate and the absorbance was measured at 420 nm.
Azocasein substrate
Manufactured by Merck Group
Sourced in United States
Azocasein substrate is a laboratory reagent used for the quantitative determination of proteolytic enzyme activity. It consists of casein, a milk-derived protein, that has been chemically modified to contain an azo dye. When proteolytic enzymes act on the azocasein substrate, the dye is released, allowing for colorimetric measurement of enzyme activity.
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Lab products found in correlation
2 protocols using azocasein substrate
1
Azocasein Assay for Protease Activity
2
Azocasein-Based Proteolytic Assay Protocol
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Azocasein substrate (Sigma Chemical Co., St Louis, USA) was used for proteolytic assay (Elpidina et al. 2001 (link)) with minor modification. The reaction mixture containing 80 μl of 1.5% azocasein solution in sodium phosphate-borate buffer (0.05 M, pH 12) was mixed with 50 μl of the enzyme extract and incubated at 37 °C for 50 min. The proteolysis was terminated by the addition of 100 μl 30% trichloroacetic acid (TCA) as a stopper, and then rested at 4 °C for 30 min. Precipitations were reached by centrifugation of 13,000 rpm for 10 min at 4 °C. Then 100 μl of supernatant was mixed with 100 μl of 2 M NaOH, and the absorbance was read at 440 nm. In the blanks, TCA was added to the mixture before adding the enzyme extract. All experiments were carried out in three replicates, and each experiment was repeated at least three times.
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