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Technical Data Sheet

BK6950

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Bad Phospho-Regulation
Antibody Sampler Kit
Price

$395

Kit Components
Applications: WB = Western blot, E = ELISA, ICC = Immunocytochemistry, IP = Immunoprecipitation, IHC = Immunohistochemistry Species: H = Human, R = Rat, M = Mouse, C = Chicken, F = Fish

Product References
RS3251 Kawasaki, H. et al. (2013) World J Gastroenter. 19(17):2629. WB, ICC: mouse intestinal myofibroblasts
MS3001 Estrada-Bernal, A. et al. (2011) J Neurooncol. 102:353. Western Blot
RS3251 Estrada-Bernal, A. et al. (2011) J Neurooncol. 102:353. Western Blot
Kit Summary
The Bad phospho-regulation antibody sampler kit can be used to detect phosphorylation of Ser-26 and Ser-112 relative to total Bad expression levels. The kit includes rabbit polyclonal phospho-specific antibodies to Ser-26 and Ser-112, as well as a mouse monoclonal antibody for detection of total Bad. The kit also includes anti-Rabbit Light Chain specific:HRP and anti-Mouse Ig:HRP secondary reagents for detection of antibodies in western blot, ELISA, or immunocytochemistry.
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Enlarge

Western blot analysis of mouse J774A.1 macrophage stimulated with calyculin A (lanes 1-4) then dephosphorylated with lambda phosphatase (lanes 2 & 4). The blots were probed with mouse monoclonal anti-Bad (lanes 1 & 2), and rabbit polyclonal anti-Bad (Ser-26) (lanes 3 & 4).

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Enlarge

Western blot analysis of mouse J774A.1 macrophage stimulated with calyculin A (lanes 1-4) then dephosphorylated with lambda phosphatase (lanes 2 & 4). The blots were probed with mouse monoclonal anti-Bad (lanes 1 & 2), and rabbit polyclonal anti-Bad (Ser-112) (lanes 3 & 4).

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Background
Bad is a member of the BCL-2 family of regulators involved in 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 IKK, and MAP kinases, as well as protein phosphatase calcineurin are found to be involved in the regulation of this Bad activity. Phosphorylation of Bad occurs on one or more of Ser-26, Ser-112, Ser-136, and Ser-155 in response to survival stimuli, which blocks its pro-apoptotic activity. Phosphorylation on Ser-136 or Ser-112 promotes heterodimerization with 14-3-3 proteins. This interaction then facilitates the phosphorylation at Ser-155, a site within the BH3 motif, leading to the release of Bcl-xL and the promotion of cell survival. Ser-26 is phosphorylated by IKK leading to phosphorylation of C-terminal serine sites and disruption of binding to Bcl-xL. This inactivation of Bad inhibits TNFα-induced apoptosis independent of NF-κB activity.

Background References
Danial, N.N. (2008) Oncogene. 27(Suppl.): S53.
Yan, J. et al. (2013) Cell. 152:304.
Buffer and Storage
Primary antibodies are supplied in phosphate-buffered saline, 50% glycerol, 1 mg/ml BSA, and 0.05% sodium azide. The secondary reagents are supplied in the same buffer without azide. BP4971 is supplied in phosphate-buffered saline and 0.09% sodium azide. Store all at –20°C. Stable for 1 year.
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