Cell Signaling Technology

Product Pathways - Apoptosis / Autophagy

Phospho-Bad Antibody Sampler Kit #9105

Kit Includes Quantity Applications Reactivity MW (kDa) Isotype
Bad (D24A9) Rabbit mAb #9239 40 µl W H M R Mk 23 Rabbit IgG
Phospho-Bad (Ser112) (40A9) Rabbit mAb #5284 40 µl W IHC-P F H M R Mk 23 Rabbit IgG
Phospho-Bad (Ser136) (185D10) Rabbit mAb #5286 80 µl W M Mk (H) (R) 23 Rabbit IgG
Phospho-Bad (Ser155) Antibody #9297 40 µl W M (H) (R) 23 Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
pCMV-Tag4A-mBad/GrpE 20 micrograms

Applications Key:  W=Western Blotting  IHC-P=Immunohistochemistry (Paraffin)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Bad (D24A9) Rabbit mAb detects endogenous levels of total Bad protein. Phospho-Bad (Ser112) (40A9) Rabbit mAb detects endogenous levels of Bad only when phosphorylated at Ser112. The Ser112 nomenclature is based upon the mouse sequence. The analogous phosphorylation sites are Ser75 in human and Ser113 in rat. Phospho-Bad (Ser136) (185D10) Rabbit mAb detects immunoprecipitated or transfected levels of Bad protein only when phosphorylated at Ser136. The Ser136 nomenclature is based upon the mouse sequence. The analogous phosphorylation sites are Ser99 in human and Ser137 in rat. Phospho-Bad (Ser155) Antibody detects transfected levels of Bad only when phosphorylated at Ser155. The Ser155 nomenclature is based upon the mouse sequence. The analogous phosphorylation sites are Ser118 in human and Ser156 in rat.

Western Blotting

Western Blotting

Western blot analysis of extracts from COS cells, untreated or TPA-treated, using Phospho-Bad (Ser112) (40A9) Rabbit mAb #5284 (upper) or Bad Antibody #9292 (lower).

Western Blotting

Western Blotting

Western blot analysis of extracts from control 293, GST-Bad transfected 293 and COS cells, untreated or TPA-treated, using Phospho-Bad (Ser136) (185D10) Rabbit mAb #5286.

Western Blotting

Western Blotting

Western blot analysis of HeLa cells, mock transfected or transfected with pCMV-Tag4A-mBad/GrpE, using Bad (D24A9) Rabbit mAb #9239.


Western Blotting

Western Blotting

Western blot analysis of HeLa cells, mock transfected or transfected with pCMV-Tag4A-mBad/GrpE, untreated or TPA- and/or λ-phosphatase-treated, using phospho- and total Bad antibodies #5286, #9296, #9297, and #9268. Note that overexpressed fusion protein runs as a 48 to 52 kDa doublet that is likely formed as a result of alternative start sites.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Bad (D24A9) Rabbit mAb #9239.

Western Blotting

Western Blotting

Western blot analysis of cell extracts from 293 cells transfected with Wild-type Bad, Bad (S112A/S136A), Bad (S155A) and treated with forskolin (30 µM), using Phospho-Bad (Ser155) Antibody #9297 (upper) and Bad Antibody #9292 (lower).


Description

The Phospho-Bad Antibody Sampler Kit provides an economical means to investigate the role of Bad protein in apoptosis. The kit contains primary and secondary antibodies to perform four Western blots with each antibody.

Source / Purification

Bad (D24A9) Rabbit mAb (#9239) was prepared by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro102 of human Bad. Phospho-specific antibodies (#5284, 5286, 9297) were prepared by respectively immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser112, Ser136, Ser155 of mouse Bad. Polyclonal Phospho-Bad (Ser155) Antibody (#9297) was purified by protein A and peptide affinity chromatography. The construct pCMV-Tag4A-mBad/GrpE expresses a fusion protein of mouse Bad with the E. coli heat shock protein GrpE. The fusion protein runs as a 48 to 52 kDa doublet formed by alternative start sites.

Background

Bad is a proapoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL (1,2). Survival factors, such as IL-3, inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (2). Phosphorylation at these sites promotes binding of Bad to 14-3-3 proteins to prevent an association between Bad with Bcl-2 and Bcl-xl (2). Akt phosphorylates Bad at Ser136 to promote cell survival (3,4). Bad is phosphorylated at Ser112 both in vivo and in vitro by p90RSK (5,6) and mitochondria-anchored PKA (7). Phosphorylation of Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10).

  1. Yang, E. et al. (1995) Cell 80, 285-291.
  2. Zha, J. et al. (1996) Cell 87, 619-628.
  3. Datta, S.R. et al. (1997) Cell 91, 231-241.
  4. Peso, L. et al. (1997) Science 278, 687-689.
  5. Bonni, A. et al. (1999) Science 286, 1358-1362.
  6. Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
  7. Harada, H. et al. (1999) Mol. Cell 3, 413-422.
  8. Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
  9. Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
  10. Datta, S. et al. (2000) Mol. Cell 6, 41-51.

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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