Nupage mops sds buffer kit

Protein was isolated from harvested cells using mechanical disruption and the Mammalian Cell Lysis Kit (Sigma-Aldrich, St Louis, MO) according to the manufacturer’s instruction. The proteins were separated on 1 mm NuPage Novex 10% Bis-Tris gels using the NuPage MOPS SDS Buffer Kit (Life Technologies, Carlsbad, CA, USA) followed by electrotransfer to 0.2 mm nitrocellulose membranes (Pall, Port Washington, WI, USA). Nonspecific binding sites were blocked with 5% bovine serum albumin in PBS for 1 hour at room temperature (RT). Membranes were then incubated with diluted primary antibody (1:1000; Cell Signaling Technology, Inc.) at 4°C overnight. Following three washes with PBS containing 0.5% Tween-20, membranes were incubated with diluted secondary antibody (GE Healthcare, Buckinghamshire, UK) at RT for two hours. The signal was visualized with an enhanced chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ). For the protein loading control, blots were stripped and stained for GAPDH using an anti-GAPDH antibody (1:2000, AbCam, Cambridge, MA).

Proteins were isolated from harvested cells by mechanical disruption and the Mammalian Cell Lysis Kit (Sigma) according to the manufacturer’s instruction. The proteins were separated on 1-mm NuPage Novex 10% Bis-Tris gels using the NuPage MOPS SDS Buffer Kit (Life Technologies, Carlsbad, CA, USA) followed by electrotransfer to 0.2-mm nitrocellulose membranes (Pall, Port Washington, WI, USA). Nonspecific binding sites were blocked with 5% bovine serum albumin in PBS for 1 h at room temperature. The membranes were then incubated with diluted FGFR2 (1:1000; Cell Signaling Technology, Inc.) at 4 °C overnight. After three washes with PBS containing 0.5% Tween-20, the membranes were incubated with a diluted horseradish peroxidase-conjugated secondary anti-rabbit (GE Healthcare, Buckinghamshire, UK) at room temperature for 2 h. The signal was visualized with enhanced chemiluminescent reagent (Amersham Biosciences, Piscataway, NJ, USA). As a protein loading control, blots were stripped and stained for GAPDH using an anti-GAPDH antibody (1:2000, AbCam, Cambridge, MA, USA).