Nupage novex bis tris midi gels

Melanoma protein cell lysates were separated using SDS page using 4%–12% NuPAGE® Novex Bis‐Tris Midi‐ Gels (Invitrogen, Carlsbad, CA), and then transferred to a nitrocellulose membrane using iBot2 dry blotting system (Invitrogen, Carlsbad, CA). The membranes were then blocked using 5% BSA for 1 h, before incubation with primary antibody at 4°C overnight. To remove residual primary antibodies the membranes were washed for 3 × 10 min in TBS/T (20 mM Tris–HCl pH 7.5, 137 mM NaCl, and 0.1% tween20). Next, a horseradish peroxidase conjugated secondary antibody was applied for 1 h at room temperature (Dako, Glostrup, Denmark). The protein bands were visualized by chemiluminescence using SuperSignal™ West Dura Extended Duration substrate (Thermo Fisher Scientific, Waltham, MA, USA). Protein bands were visualized using the Bio‐Rad ChemiDoc™ imaging system (Bio‐Rad), and images were prepared using Adobe Photoshop CC 2017 (San Jose, CA, USA).

Preparations of whole cell lysates were performed by addition of lysis buffer (150mM NaCl, 50mM Tris-HCl pH 7.5, 0.1% Nondient P-40, 1x Complete tablets mini EASYpack and 1x PhosSTOP from Roche), to frozen, dry cell pellets. The samples were left on ice for 30min with intermittent vortexing, followed by sonication and centrifugation to remove cell debris. Protein quantification was done using a standard Bradford protein assay (Bio-Rad Laboratories, Inc., CA). Protein lysates (30μg) were separated on 4-12% NuPAGE® Novex Bis-Tris Midi-Gels (Invitrogen, Carlsbad, CA) and then transferred to a membrane using an iBlot Dry Blotting system (Invitrogen, Carlsbad, CA) according to the manufacturers instruction. The membranes were subsequently blocked in TBS/T (137mM NaCl, 20mM Tris-HCl pH 7.5 and 0.1% tween20) containing 5% BSA (Sigma-Aldrich, Steinheim, Germany) for 1h, and then incubated with primary antibodies over night at 4°C with gentle agitation. Residual primary antibodies were removed by washing 3 × 10min in TBS/T. The membranes were thereafter incubated for 1h at RT with horse radish peroxidase conjugated (HRP) secondary antibody. The membranes were then washed 3 × 10min with TBS/T. The protein bands were visualized in the G-Box iChemi Chemiluminescence Image Capture (Syngene, England) using the SuperSignal chemiluminescence detection system (Pierce).