Anti mouse igg conjugated with horseradish peroxidase

To prepare crude membrane fractions, transformed T87 cells were harvested on a 40 lm nylon mesh (Millipore, Billerica, MA, USA) by filtration, frozen by liquid nitrogen and homogenized in liquid nitrogen using a mortar and pestle. The lysed cells were suspended in a 10-fold volume of extraction buffer containing 50 mM Tris/HCl (pH 8.0), 2 mM EDTA, 20% (v/v) glycerol, 1 mM dithiothreitol and 29 Complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The suspension was centrifuged at 10 000 g for 10 min at 4 °C to remove any cell debris. After centrifugation of the supernatant at 146 000 g for 40 min at 4 °C, the precipitate obtained was resuspended in extraction buffer. SDS/ PAGE and immunoblotting were carried out as described previously with the modifications as described below [34] .
The membrane was incubated with monoclonal anti-HA Ig (Nacalai Tesque, Kyoto, Japan) at a dilution of 1 : 40 000 and then with anti-mouse IgG conjugated with horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA) at a dilution of 1 : 200 000 in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Osaka, Japan). A SuperSignal West Femto chemiluminescent kit (Thermo Scientific, Rockford, IL, USA) was used to detect antigens.