Hrp conjugated anti mouse igg secondary antibody

Cells were washed with PBS and harvested in RIPA buffer containing protease and phosphatase inhibitors. Lysates were quantified by the Bradford method using BSA as a standard. The proteins were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% BSA for 2h at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-FGFR1 (1:500, ab76464), anti-pFGFR1 (1:750, ab59194), and anti-V5 (1:1500, ab27671). Beta-tubulin (1:2000, ab6046) was used as loading control. Membranes were washed with TBST and then incubated with HRP-conjugated anti-rabbit IgG secondary antibody (1:5000, NA934V) or HRP-conjugated anti-mouse IgG secondary antibody (1:5000; NB7539) for 1h at room temperature and revealed utilizing Amersham ECL detection (Amersham Biosciences).