Mes or mops sds running buffer

Cells grown in six-well plate were washed twice with PBS and solubilized with 2% CHAPS buffer (25 mM HEPES-KOH pH 7.5, 300 mM NaCl, 2% (w/v) CHAPS, protease inhibitor cocktail (Roche, Indianapolis, IN)) on ice for 30 min and then protein concentrations were determined. Proteins precipitated with TCA were lysed with SDS-PAGE sample buffer supplemented with DTT. The appropriate amounts of proteins were applied and separated on 4–12% Bis-Tris SDS-PAGE (Invitrogen) with MES or MOPS SDS running buffer (Invitrogen). After transfer, PVDF membrane were blocked and incubated with primary antibodies. Proteins were detected using alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG as secondary antibodies and a BCIP-NBT solution kit (Nacalai Tesque, Japan). For detecting phosphorylated ubiquitin, anti-rabbit IgG horseradish peroxidase-linked secondary antibodies (GE Healthcare Life Sciences) and Western Lightning Plus-ECL (PerkinElmer) were used.

SDS-PAGE was performed according to the method of Laemmli (1970) using pre-casted NuPAGE 4–12% Bis-Tris gels (Invitrogen) and MES or MOPS SDS running buffer (Invitrogen) (28 (link)). Proteins were transferred to the Hybond-P polyvinylidene fluoride membrane (GE Healthcare) (29 (link)). The membrane was blocked in 5% non-fat dried milk and 0.1% HSA, and incubated with primary monoclonal antibodies (0.5 µg/ml). Subsequently, the membrane was washed and incubated with HRP-conjugated rabbit anti-mouse antibody diluted (1:20,000) accordingly to the manufacturer’s recommendation (Dako, Denmark) and developed by means of the ECL plus Western blotting detection kit (GE Healthcare). For specificity testing of applied antibodies, 1 µl of serum was applied to the gel per 4 mm well width.

For preparation of total cell lysate, cells grown in a six-well plate were washed twice with PBS and solubilized with 2% CHAPS buffer (25 mM HEPES-KOH pH 7.5, 300 mM NaCl, 2% (wt/vol) CHAPS, protease inhibitor cocktail (Roche, Indianapolis, IN) on ice for 30 min and then protein concentrations were determined. Proteins precipitated with trichroloacetic acid were lysed with NuPAGE LDS sample buffer (Invitrogen) supplemented with 80 mM dithiothreitol. The appropriate amounts of proteins were applied and separated on 4–12% Bis-Tris SDS-PAGE (Invitrogen) with MES or MOPS SDS running buffer (Invitrogen). After transfer to PVDF membrane, blocking and incubation with primary antibodies, proteins were detected using horseradish peroxidase-coupled secondary antibodies (GE Healthcare Life Sciences, Piscataway, NJ) and ECL Plus or ECL Prime western blotting detection reagents (GE Healthcare Life Sciences).