Product Pathways - Apoptosis / Autophagy

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Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit #9942

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
Bax (D2E11) Rabbit mAb #5023	40 µl	W IP IHC-P	H	20	Rabbit IgG
Bik Antibody #4592	40 µl	W IHC-P	H	20	Rabbit
Bim (C34C5) Rabbit mAb #2933	40 µl	W IP IHC-P IF-IC F	H M R (Mk) (B) (Dg)	12,15,23	Rabbit IgG
BID Antibody (Human Specific) #2002	40 µl	W IP	H	15, 22	Rabbit
Bak (D2D3) Rabbit mAb #6947	40 µl	W IP IHC-P	H Mk	25	Rabbit IgG
Puma Antibody #4976	40 µl	W	H (Mk)	23	Rabbit
Anti-rabbit IgG, HRP-linked Antibody #7074	100 µl				Goat

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

Specificity / Sensitivity

Each antibody in the Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit recognizes only its specific target. The antibodies do not cross-react with other Bcl-2 family members. Phospho-Bad (Ser112) (40A9) Rabbit mAb detects endogenous levels of Bad only when phosphorylated at Ser112 (mouse), Ser75 (human), or Ser113 (rat).

Description

The Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit provides an economical means to examine several members of the Bcl-2 family and their activation status. The kit contains enough primary and secondary antibodies to perform four Western blot experiments per primary antibody.

Source / Purification

Phospho-Bad (Ser112) (40A9) Rabbit mAb is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser112 of mouse Bad. Total Bad, Bax, Bim, and Bak monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Pro102 of human Bad, Leu45 of human Bax, Pro25 of human Bim, or Gly82 of human Bak. Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to the amino-terminus of human Bik; residues surrounding the cleavage site of human BID; or the carboxy terminus of human Puma. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

The Bcl-2 family consists of a number of evolutionarily conserved proteins containing Bcl-2 homology domains (BH) that regulate apoptosis through control of mitochondrial membrane permeability and release of cytochrome c (1-3). Four BH domains have been identified (BH1-4) that mediate protein interactions. The family can be separated into three groups based upon function and sequence homology: pro-survival members include Bcl-2, Bcl-xL, Mcl-1, A1 and Bcl-w; pro-apoptotic proteins include Bax, Bak and Bok, and "BH3 only" proteins Bad, Bik, Bid, Puma, Bim, Bmf, Noxa and Hrk. Interactions between death-promoting and death-suppressing Bcl-2 family members has led to a rheostat model in which the ratio of pro-apoptotic and anti-apoptotic proteins controls cell fate (4). Thus, pro-survival members exert their behavior by binding to and antagonizing death-promoting members. In general, the "BH3-only members" can bind to and antagonize the pro-survival proteins leading to increased apoptosis (5). While some redundancy of this system likely exists, tissue specificity, transcriptional and post-translational regulation of many of these family members can account for distinct physiological roles.

Bad is a pro-apoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death (6,7). Survival factors such as IL-3 can inhibit the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136 (7). Phosphorylation at these sites results in the binding of Bad to 14-3-3 proteins and the inhibition of Bad binding to Bcl-2 and Bcl-xL (7). Akt has been shown to promote cell survival via its ability to phosphorylate Bad at Ser136 (8,9). Ser112 has been shown to be the substrate in vivo and in vitro of p90RSK (10,11) and mitochondria-anchored PKA (12).

1. Cory, S. et al. (2003) Oncogene 22, 8590-607.
2. Antonsson, B. and Martinou, J.C. (2000) Exp Cell Res 256, 50-7.
3. Sharpe, J.C. et al. (2004) Biochim Biophys Acta 1644, 107-13.
4. Korsmeyer, S.J. et al. (1993) Semin Cancer Biol 4, 327-32.
5. Bouillet, P. and Strasser, A. (2002) J Cell Sci 115, 1567-74.
6. Yang, E. et al. (1995) Cell 80, 285-291.
7. Zha, J. et al. (1996) Cell 87, 619-628.
8. Datta, S. R. et al. (1997) Cell 91, 231-241.
9. Peso, L. et al. (1997) Science 278, 687-689.
10. Bonni, A. et al. (1999) Science 286, 1358-1362.
11. Tan, Y. et al. (1999) J. Bio. Chem. 274, 34859-34867.
12. Harada, H. et al. (1999) Mol. Cell 3, 413-422.

Application References

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Protocols

2002:

Immunoprecipitation, Western Blotting

2933:

Flow, IHC / Paraffin, Immunofluorescence, Immunoprecipitation, Western Blotting

4592:

IHC / Paraffin, Western Blotting

4976:

Western Blotting

5023:

IHC / Paraffin, Immunoprecipitation, Western Blotting

5284:

Flow, IHC / Paraffin, Western Blotting

6947:

IHC / Paraffin, Immunoprecipitation, Western Blotting

9239:

Western Blotting

Companion Products

• 9941 Pro-Survival Bcl-2 Family Antibody Sampler Kit
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide

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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.