Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

Cells were lysed in lysis buffer (50 mM HEPES-pH7.5, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.5% triton X-100, 10% glycerol) on ice for 10 min. Clear cell lysates were collected after centrifugation and protein concentration was determined by Bradford assay (BioRad). Fifteen micrograms of total protein from each treatment was loaded and separated by 12% SDS-PAGE electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane (PVDF; PerkinElmer) by a Trans-Blot semidry blotting system (BioRad). The membrane was incubated with primary rabbit anti-GFP polyclonal antibody (1:1000 dilution; BioVision) or with primary mouse anti-␤-actin monoclonal antibody (1:5000 dilution; Abcam) at room temperature for 1 h after 5% skim milk blocking. It was then reacted with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG, 1:10 000 dilution; Jackson). The blotting signals were visualized by Western lighting plus ECL (PerkinElmer) and detected by an ImageQuant TM LAS-4000 mini luminescent image analyzer (GE).