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Technical Data Sheet

BP4951

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Bad (Ser-26), phospho-specific
Rabbit Polyclonal
Price
Size
Species Reactivity
MW

$295
100 μl
Rt, Ms
23 kDa

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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).

Application
Dilution


ELISA
1:1000


WB
1:500



End user should determine optimal dilution for their particular applications and experiments.Western blot membranes were incubated with diluted antibody in 5% non-fat milk, PBS, 0.04% Tween20 for 1hour at room temperature.
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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.
Immunogen
Phospho-Bad (Ser-26) synthetic peptide (coupled to KLH) corresponding to amino acid residues surrounding serine 26 in mouse Bad. This peptide sequence is highly conserved in rat Bad, but is not found in human Bad.
Buffer and Storage
Rabbit polyclonal, affinity-purified antibody is supplied in 100µl phosphate-buffered saline, 50% glycerol, 1 mg/ml BSA, and 0.05% sodium azide. Store at –20°C. Stable for 1 year.
Specificity
This antibody was affinity purified using phospho-Bad (Ser-26) peptide (without carrier). The antibody detects a 23 kDa* doublet corresponding to the apparent molecular mass of phosphorylated Bad on SDS-PAGE immunoblots of mouse J774A.1 treated with calyculin A. This reactivity is not observed after lambda phosphatase treatment.

*All molecular weights (MW) are confirmed by comparison to Bio-Rad Rainbow Markers and to western blot mobilities of known proteins with similar MW.
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