Mini protean tgx pre cast stain free gels

The puromycin incorporation assay was performed as described in Panas et al. (2015b) (link) with the following modifications. Puromycin was added to the medium at the final concentration of 10 µg/ml for 10 min. Cells were washed with PBS and the whole-cell lysate preparation and western blotting analysis were done as described above. For electrophoresis, samples were loaded onto Mini-PROTEAN TGX Pre-cast Stain-Free gels (5-15%, Bio-Rad Laboratories, Hercules, CA, USA) and total protein was visualised post-transfer to PVDF membranes on ChemiDoc Touch Imaging System. Puromycin incorporation into nascent polypeptides was visualised using anti-puromycin antibody (1:6000; mouse, MilliporeSigma, MABE343), quantified using Bio-Rad Image Lab 5.2.1 software and values normalised to the StainFree signal for each lane.

The puromycin incorporation assay was performed as described in [106 (link)] with the following modifications. Puromycin was added to the medium at the final concentration of 10 μg/ml for 10 min. Cells were washed with PBS and the whole-cell lysate preparation and western blotting analysis were done as described above. For electrophoresis, samples were loaded onto Mini-PROTEAN TGX Pre-cast Stain-Free gels (5–15%, BioRad Laboratories, Hercules, CA, USA) and total protein was visualised post-transfer to PVDF membranes on ChemiDoc Touch Imaging System. Puromycin incorporation into nascent polypeptides was visualised using anti-puromycin antibody (1:6,000; mouse, MilliporeSigma, MABE343).