Product Pathways - Apoptosis
Phospho-Bad Antibody Sampler Kit #9105
|9105S||1 Kit (4 x 40 µl)||---||In Stock||---|
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|Kit Includes||Quantity||Applications||Reactivity||Homology†||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|
†Species predicted to react based on 100% sequence homology.
Applications Key: W=Western Blotting, IHC-P=Immunohistochemistry (Paraffin), F=Flow Cytometry
Reactivity Key: H=Human, M=Mouse, R=Rat, Mk=Monkey
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 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 blot analysis of HeLa cells, mock transfected or transfected with pCMV-Tag4A-mBad/GrpE, using Bad (D24A9) Rabbit mAb #9239.
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 blot analysis of extracts from various cell lines using Bad (D24A9) Rabbit mAb #9239.
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).
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.
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.
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.
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 at Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL (8-10).
- Yang, E. et al. (1995) Cell 80, 285-291.
- Zha, J. et al. (1996) Cell 87, 619-628.
- Datta, S.R. et al. (1997) Cell 91, 231-241.
- Peso, L. et al. (1997) Science 278, 687-689.
- Bonni, A. et al. (1999) Science 286, 1358-1362.
- Tan, Y. et al. (1999) J. Biol. Chem. 274, 34859-34867.
- Harada, H. et al. (1999) Mol. Cell 3, 413-422.
- Tan, Y. et al. (2000) J. Biol. Chem. 275, 25865-25869.
- Lizcano, J. et al. (2000) Biochem. J. 349, 547-557.
- Datta, S. et al. (2000) Mol. Cell 6, 41-51.
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- 7072 Phototope®-HRP Western Blot Detection System, Anti-mouse IgG, HRP-linked Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
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- 7727 Biotinylated Protein Ladder Detection Pack
- 7003 20X LumiGLO® Reagent and 20X Peroxide
For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
Select rabbit monoclonal antibodies are developed, validated, and produced at CST using in part technology under license (granting certain rights including those under U.S. Patents No. 5,675,063 and in some instances 7,429,487) from Epitomics, Inc.