2.7. The in‐gel activity assays

Cells (1 × 10⁵) were plated in 35 mm NUNC plate, and after 6 hours, the complete medium was replaced with serum‐free medium and incubated further for 24 hours. An in‐house procedure used to remove albumin from the medium, and the conditioned medium was subjected to freeze dry. For the in‐gel assay, 12% SDS‐polyacrylamide gel (SDS‐PAGE) was prepared with Bovine β‐casein (0.5 mg/mL; C6905‐1G, Sigma‐Aldrich). The pre‐run was done at room temperature at a constant current (40 mA) for 30 minutes. The Laemilli buffer (1×) reducing agents was added to the sample (30 μL), boiled, and loaded on to the gel. The electrophoresis was performed at constant current (20 mA at 4°C). The gel was washed twice with 2.5% Triton X‐100 and 50 mM Tris‐Cl (pH 7.5). Then the gel was washed twice with 50 mM Tris‐Cl buffer, for 10 minutes and incubated at 37°C overnight in developing buffer (0.15 M NaCl, 10 mM CaCl₂, 0.1% Triton X‐100, 0.02% NaN₃, 50 mM Tris‐Cl, pH 7.5). Subsequently, the gel was stained (with 0.5% Coomassie Brilliant Blue R 250 in 10% acetic acid), destained with 10% acetic acid until the clean bands were visualized.

To the 30 μL sample prepared from the conditioned medium, Laemilli buffer (1×) reducing agents was added the sample (30 μL), boiled, and loaded on to the gel. SDS‐polyacrylamide gel (10%) was polymerized with 0.1% gelatin (G9136‐10MG, Sigma‐Aldrich). The gel run was performed at a constant voltage (100 V) for one and a half hours. Then the gel was washed with (2.5%, ×2) Triton X‐100 for 30 minutes to remove SDS. Further, the gel was incubated at 37°C for overnight in the developing buffer (0.15 M NaCl, 10 mM CaCl₂, 0.5 mM ZnCl₂, 50 mM Tris‐Cl, pH 7.5). The gel was stained (with 0.5% Coomassie Brilliant Blue R 250 in 10% acetic acid), destained with 10% acetic acid until the clean bands were visualized.