Avidin hrp

Levels of intercellular BMP-4 and that in conditioned medium were determined by ELISA using mouse anti-human BMP-4 (R&D) as the primary antibody, with biotinylated sheep anti-mouse IgG (Amersham) and avidin-HRP (Boehringer) utilized as secondary antibodies.

P19 cells were cultured in bacterial-grade dishes in the presence or absence of RA for 24 h. Then, the cells were collected and washed with PBS, resuspended in 0.34 M sucrose solution (0.34 M sucrose, 20 mM Tris–HCl [pH 7.4], 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, Proteinase Inhibitor Cocktail [III]). Cell suspensions were homogenized using a Teflon homogenizer on ice, and then the homogenates were centrifuged at 700×g for 10 min at 4 °C to remove the nuclear fraction, after which the supernatants were centrifuged at 1000×g for 30 min at 4 °C. The supernatants were used as the cytosol fraction, and the pellets were dissolved with TBS containing 0.5 % Triton X-100 as the mitochondrial fraction. The amount of cytoplasmic cytochrome c in these fractions was measured by sandwich ELISA-based method using a Cytochrome c Mouse/Rat ELISA Quantikine kit (R&D Systems) according to the manufacturer’s instructions. The amounts of cytochrome c in both the mitochondrial and cytoplasmic fractions were used to determine the ratio of cytoplasmic fractionation. The expression of Bcl family proteins was measured by ELISA using anti-Bak antibody (G-23), anti-Bax antibody (N-20), BCL-XL antibody (7D9), and anti-Bcl-xL antibody (H-5) (Santa Cruz Biotechnology) as primary antibodies, and biotinylated goat-anti-rabbit IgG as the secondary antibody with avidin-HRP (Boehringer).