Immunofluorescent analysis of HeLa cells using a Phospho-Bad pSer99 polyclonal antibody (Product # PA5-12553). HeLa cells were fixed with 4% PFA (20 min), permeabilized with Triton X-100 (0.2%, 30 min). Cells were then incubated with a Phospho-Bad pSer99 polyclonal antibody (Product # PA5-12553) (1:200, 2 hr at room temperature). Primary antibody detected by fluor-conjugated donkey anti-rabbit secondary antibody (green) was used (1:1000, 1hr). Nuclei were counterstained with Hoechst 33342 (blue) (10 ug/ml, 5 min).
|Tested species reactivity||Human|
|Host / Isotype||Rabbit / IgG|
|Immunogen||KLH conjugated synthetic phosphopeptide corresponding to amino acid residues surrounding S99 of human Bad|
|Purification||Antigen affinity chromatography|
|Contains||0.09% sodium azide|
|Storage Conditions||Maintain refrigerated at 2-8°C for up to 6 months. For long term storage store at -20°C|
|Tested Applications||Dilution *|
|Dot blot (DB)||1:500|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
This antibody is predicted to react with mouse and rat based on sequence homology.
Bad is a member of the BCL-2 family. BCL-2 family members are known to be regulators of programmed cell death. This protein positively regulates cell apoptosis by forming heterodimers with BCL-xL and BCL-2, and reversing their death repressor activity. Proapoptotic activity of this protein is regulated through its phosphorylation. Protein kinases AKT and MAP kinase, as well as protein phosphatase calcineurin are found to be involved in the regulation of this protein. Bad is phosphorylated on one or more of Ser-75, Ser-99, Ser-118 and Ser-134 in response to survival stimuli, which blocks its pro-apoptotic activity. Phosphorylation on Ser-99 or Ser-75 promotes heterodimerization with 14-3-3 proteins. This interaction then facilitates the phosphorylation at Ser-118, a site within the BH3 motif, leading to the release of Bcl-X(L) and the promotion of cell survival. Ser-99 is the major site of AKT/PKB phosphorylation, Ser-118 the major site of protein kinase A (CAPK) phosphorylation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.