Super signal west dura extended duration subtract kit

Transfected Bm5 and High Five cells were collected and lysed by sonication (Branson) in a lysis buffer consisting of Tris-Cl 50 mM, NaCl 150 mM, EDTA 1 mM, Triton-X-100 1%, Sodium Dodecyl Sulphate (SDS) 0.5% and Protease Inhibitor Cocktail Tablets Complete (Roche). Next, the total amount of proteins was quantified by means of a Bicinchoninic acid assay, after which 18 μg of each sample were separated by SDS-polyacrylamide gel electrophoresis. Then, the proteins were transferred to a Trans-Blot Turbo Mini PVDF membrane using the Trans-Blot Turb Blotting System (Bio-Rad). The blots were washed and blocked with a skimmed powder milk solution 5% for 2 hours. Anti-c-Myc antibodies produced in rabbit (Sigma-Aldrich) were diluted (1:5000) and incubated with the blots overnight at room temperature. Washing was then performed, followed by 2 hours of incubation with Polyclonal Goat Anti-Rabbit Immunoglobulins/HRP (Dako), diluted 1:50000. Finally, the blots were washed and the detection was performed with Super Signal West Dura Extended Duration Subtract Kit (Thermo Scientific). The chemiluminescent bands were visualized using a ChemiDoc™ MP Imaging System with Image Lab Software (Bio-Rad).

Transfected Bm5 and High Five cells were collected and lysed by sonication (Branson) in a lysis buffer consisting of Tris-Cl 50 mM, NaCl 150 mM, EDTA 1 mM, Triton-X-100 1%, sodium dodecyl sulphate (SDS) 0.5% and Protease Inhibitor Cocktail Tablets Complete (Roche, Basel, Switzerland). Next, the total amount of proteins was quantified by means of a bicinchoninic acid assay, after which 18 μg of each sample were separated by SDS-polyacrylamide gel electrophoresis. Then, the proteins were transferred to a Trans-Blot Turbo Mini PVDF membrane using the Trans-Blot Turbo Blotting System (Bio-Rad, Hercules, CA, USA). The blots were washed and blocked with a 5% skimmed powder milk solution for 2 h. Mouse anti-c-Myc (Cell Signalling, Danvers, MA, USA) and rabbit anti-Flag antibodies (Sigma-Aldrich, Bornem, Belgium) were diluted (1:5000) and incubated with the blots overnight at room temperature. Washing was then performed, followed by 2 h of incubation with Polyclonal Goat Anti-Mouse or Anti-Rabbit Immunoglobulins/HRP (Dako), diluted 1:50,000. Finally, the blots were washed, and the detection was performed with the Super Signal West Dura Extended Duration Subtract kit (Thermo Scientific, Waltham, MA, USA). The chemiluminescent bands were visualized using a ChemiDoc^™ MP Imaging System with Image Lab Software (Bio-Rad, Hercules, CA, USA).