Xcell 2 blot module for wet tank transfer

10 μL of culture supernatant (containing secreted carboxylesterase enzyme) were run on a reducing sodium dodecyl sulfate (SDS) NuPAGE® 12% Bis-Tris polyacrylamide gel (Life technologies™) with NuPAGE® morpholinepropanesulfonic acid (MOPS) buffer at 180 V for 60 min. Protein bands were visualized using Coomassie staining solution. For Western blotting, SDS-PAGE separated proteins were transferred to a nitrocellulose membrane using the XCell II™ Blot Module for wet (tank) transfer (Life technologies™) according to the manufacturer’s instructions. Carboxylesterase was detected using anti-carboxylesterase antiserum as described by Heinl et al. [48 (link)].

The SDS-PAGE was performed using sodium dodecyl sulfate (SDS) NuPAGE^® 12% Bis–Tris polyacrylamide gels (Life Technologies™) with NuPAGE^® morpholinepropanesulfonic acid (MOPS) buffer at 180 V for 60 min. Before loading the gel with 15 μL of culture supernatant, each sample supernatant was correlated to the wet cell weight of each sample using RO water.
After SDS-PAGE, the separated proteins were transferred to a nitrocellulose membrane using the XCell II™ Blot Module for wet (tank) transfer (Life technologies™) according to the manufacturer’s instructions. The HyHEL-Fab was immunologically detected with Anti-Human IgG antibody (Abcam, ab7497) produced in mouse, specifically recognizing the Hinge region of Human IgG and Anti-Mouse IgG antibody (Sigma-Aldrich, A3673) coupled with HRP, which was produced in goat. Carboxylesterase was immunologically detected with rabbit anti-CES antiserum (produced in rabbit, provided by Biomin Holding GmbH) and carboxypeptidase Y was detected using anti-CPY antiserum (produced in rabbit, kindly provided by Günther Daum/Karlheinz Grillitsch, Graz University of Technology). As secondary antibodies, anti-rabbit IgG antibodies coupled with horseradish peroxidase (produced in goat, Sigma-Aldrich, A0545) were used.