Bluesafe stain

Protein content was determined by Bradford analysis (BIORAD) according to the manufacturer’s protocol. 15 µg protein sample was mixed 1:1 with native sample buffer (BIORAD) and loaded on a Mini-PROTEAN TGX Precast Gel (BIORAD). The PAGE was run at 4°C and low voltage for 2 hr in Tris/Glycine buffer (BIORAD). The gel was rinsed with H₂O for 5 min and stained with 50 ml staining solution (15 ml 3,3',5,5'-tetramethylbenzidine (TMBZ) solution (6.3 mM TMBZ in methanol) +35 ml 0.25 M sodium acetate solution (pH 5)) for 1 hr at room temperature (RT) while gently agitating. The gel was then incubated for 30 min at RT in the dark in presence of 30 mM H₂O₂. The background staining was removed by incubating the gel in a solution of isopropanol/0.25 M sodium acetate (3:7). Following scanning, the gel was completely destained in a solution of isopropanol/0.25 M sodium acetate (3:7) and stained with the BlueSafe stain (nzytech) for 10 min.