Runblue lds sample buffer

Protein concentrations were determined using a Nanodrop 2000 instrument (Thermo Scientific). Membrane and soluble fractions were diluted to ~3 mg ml⁻¹ in 1 × RunBlue LDS sample buffer (Expedeon) with β-mercaptoethanol (5 % vol/vol), and then incubated at 37 °C for 1 h prior to separation in RunBlue 4–12 % gradient SDS protein gels (Expedeon). For protein staining, gels were soaked in InstantBlue protein stain (Expedeon). Proteins were then transferred to 0.45 µm Amersham Hybond PVDF Blotting Membrane (GE healthcare) using the XCell II Blot Module (Invitrogen). Transfer was carried out at 30 V for 1 h. The preparation of the membrane for Western blotting (washing steps and antibody addition) was carried out according to the manufacturer’s instructions for the StrepII tag antibody-HRP conjugate (Novagen). Chemiluminescent substrate for the Western blot was prepared by adding 5 ml of 100 mM Tris-HCl, pH 8.5, buffer with 0.2 mM p-coumaric acid (Sigma) and 1.25 mM luminol to 15 µl of 3 % (vol/vol) hydrogen peroxide solution. Under dark room conditions, membranes were incubated with chemiluminescent solution for 1 min. After exposure to the blot, the X-ray film (GE healthcare) was incubated for 1–3 min in developer solution (Kodak) and 30 s in fixer solution (Kodak). The film was rinsed in water and allowed to dry.