Bad, Soluble (Bcl2 Antagonist of Cell Death) Recombinant

Bad is a pro-apoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-XL, resulting in cell death (A1,2). Survival factors such as IL-3 can inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (A2). Phosphorylation at these sites results in the binding of Bad to 14-3-3 proteins and the inhibition of Bad binding to Bcl-2 and Bcl-XL (A2). Akt has been shown to promote cell survival via its ability to phosphorylate Bad at Ser136 (A3,5). Ser112 has been shown to be the substrate in vivo and in vitro of p90RSK (A6,7) and mitochondria-anchored PKA (A8). Phosphorylation of Ser155 by PKA plays a critical role in blocking the dimerization of Bad to Bcl-XL because of its position in the BH3 domain (A9-11). Source: Recombinant, full-length murine Bad fused with an N-terminal His6-tag, expressed in E. coli. Applications: Kinase Assay: Recombinant AKT1/PKBa, active, phosphorylated 10ul of soluble Bad. Subsequent immunoblot analysis detected the phosphorylated Bad using anti-phospho-Bad (Ser136). Previous experiments showed that recombinant purified AKT1/PKBa phosphorylates both S112 and S136 in vitro. Phosphorylation of BAD: 3ug of BAD was incubated with buffer alone or 250ng of active recombinant Akt1. The samples were resolved by electrophoresis, transferred to nitrocellulose and probed with a 1:1000 dilution of anti-phospho-BAD. Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system. Storage and Stability: May be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.