YOUR CART
Bad (Bcl-2 antagonist of cell death) is a proapoptotic member of the Bcl-2 family that displaces Bax from binding to Bcl-2 and Bcl-xL, which results in cell death. Bad forms heterodimers with other Bcl-2 family members and localizes to the outer mitochondrial membrane. When phosphorylated in the presence of survival signals, Bad translocates to the cytoplasm and blocks the induction of apoptosis. In the absence of survival signals, dephosphorylated Bad is localized in the outer membrane of the mitochondria where it activates mitochondrial-mediated apoptotic pathways. Thus, Bad is a unique Bcl-2 family member that plays a key role in the transduction of upstream survival signals to the Bcl-2 apoptosis decision machinery, and high-throughput study methods for monitoring Bad phosphorylation are in high demand.
Figure 1: Measurement of phosphorylated and total Bad.
COS-7 cells were cultured in 96-well plates and serum-starved for 24 hours. Cells were then treated with 200 nM of PMA for 30 minutes and fixed. Total and phospho Bad were each assayed in triplicate using the phospho and total Bad antibodies included in the FACE Bad Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).
Antibody Specificities
The phospho-Bad antibody is specific for phosphorylated Bad and was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 112 of mouse Bad. This antibody recognizes Bad only when phosphorylated at this site and does not cross-react with other sites or related family members. The total-Bad antibody recognizes Bad proteins regardless of the phosphorylation state.
