Product Pathways - Apoptosis

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Apoptotic Release Antibody Sampler Kit #8360

Kit Includes	Quantity	Applications	Reactivity	MW (kDa)	Isotype
Cytochrome c (D18C7) Rabbit mAb #11940	40 µl	W IHC-P	H M R Mk (B) (Pg)	14	Rabbit IgG
Smac/Diablo Mouse mAb #2954	40 µl	W IP IHC-P IF-IC	H Mk	21	Mouse IgG
HtrA2/Omi (D20A5) Rabbit mAb #9745	40 µl	W	H M R Mk	36	Rabbit IgG
Caspase-3 (8G10) Rabbit mAb #9665	40 µl	W IP	H M R Mk	17, 19, 35	Rabbit IgG
Cox IV (4D11-B3-E8) Mouse mAb #11967	40 µl	W IP IHC-P IF-IC	H M R Mk	17	Mouse
MEK1/2 (D1A5) Rabbit mAb #8727	40 µl	W IF-IC F	H M R Mk (Hm) Dm (X) (Z) (B) (Dg) (Pg) (Ce)	46	Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody #7074	100 µl				Goat
Anti-mouse IgG, HRP-linked Antibody #7076	100 µl				Horse

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  Hm=Hamster  Mk=Monkey  Dm=D. melanogaster  X=Xenopus  Z=Zebrafish  B=Bovine  Dg=Dog  Pg=Pig  Ce=C. elegans
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Specificity / Sensitivity

Caspase-3 (8G10) Rabbit mAb recognizes endogenous levels of full-length (35 kDa) and large fragment (17/19 kDa) caspase-3 protein resulting from cleavage at Asp175. Cox IV (4D11-B3-E8) Mouse mAb recognizes endogenous levels of total Cox IV protein. Cytochrome c (D18C7) Rabbit mAb recognizes endogenous levels of total cytochrome c protein. HtrA2/Omi (D20A5) Rabbit mAb recognizes endogenous levels of total HtrA2/Omi protein and does not cross-react with HtrA1. MEK1/2 (D1A5) Rabbit mAb recognizes endogenous levels of total MEK1 and MEK2 proteins. This antibody is predicted to cross-react with MEK1/MEK2 orthologs in a variety of species. Smac/Diablo Mouse mAb recognizes endogenous levels of processed and unprocessed human Smac/Diablo protein and reacts weakly with mouse and rat Smac/Diablo.Each antibody in the kit has been validated using the Cell Fractionation Kit #9038.

Description

The Apoptotic Release Antibody Sampler Kit provides an economical means to evaluate targets that are released from the mitochondria with apoptotic stimuli. The kit contains enough primary antibody to perform four western blots per primary antibody.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a full-length peptide corresponding to human Smac/Diablo protein raised in E. coli, or with synthetic peptides corresponding to amino-terminal residues adjacent to Asp175 of human caspase-3 protein, residues near the carboxy terminus of human Cox IV protein, residues surrounding Pro72 of human cytochrome c protein, residues surrounding Phe341 of human HtrA2/Omi protein, or residues surrounding Ala220 of human MEK1 protein.

Background

Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Initiator caspases (including 8, 9, 10, and 12) are closely coupled to proapoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6, and 7), which in turn cleave cytoskeletal and nuclear proteins like PARP, α-fodrin, DFF, and lamin A, and induce apoptosis (1).Cytochrome c is a well conserved electron-transport protein and is part of the respiratory chain localized to the mitochondrial intermembrane space (2). Upon apoptotic stimulation, cytochrome c released from mitochondria associates with procaspase-9 (47 kDa)/Apaf-1. This complex processes caspase-9 from inactive proenzyme to its active form (3). This event further triggers caspase-3 activation and eventually leads to apoptosis (4).Smac/Diablo is a 21 kDa mammalian mitochondrial protein that functions as a regulatory component during apoptosis (5,6). Upon mitochondrial stress, Smac/Diablo is released from mitochondria and competes with caspases for binding of inhibitor of apoptosis proteins (IAPs) (5,6). The interaction of Smac/Diablo with IAPs relieves the inhibitory effect of the IAPs on caspases (7,8).High temperature requirement protein A2 (HtrA2)/Omi is a serine protease with homology to the E. coli HtrA protein (DegP) and is thought to be involved in apoptosis and stress-induced degradation of misfolded proteins (9). HtrA2 is produced as a 50 kDa zymogen that is cleaved to generate a 36 kDa mature protein that exposes an amino terminal motif (AVPS) resembling that of the IAP inhibitor Smac/Diablo (10-14). Like Smac, interaction between HtrA2 and IAP family members, such as XIAP, antagonizes their inhibition of caspase activity and protection from apoptosis (10-14).Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (15). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (16).Cytochrome c oxidase (COX) is a hetero-oligomeric enzyme consisting of 13 subunits localized to the inner mitochondrial membrane (17-19). It is the terminal enzyme complex in the respiratory chain, catalyzing the reduction of molecular oxygen to water coupled to the translocation of protons across the mitochondrial inner membrane to drive ATP synthesis. The 3 largest subunits forming the catalytic core are encoded by mitochondrial DNA, while the other smaller subunits, including COX IV, are nuclear-encoded. The Cox IV (4D11-B3-E8) Mouse mAb can be used effectively as a mitochondrial loading control in cell-based research assays.MEK1 and MEK2, also called MAPK or Erk kinases, are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation (20-22). Activation of MEK1 and MEK2 occurs through phosphorylation of two serine residues at positions 217 and 221, located in the activation loop of subdomain VIII, by Raf-like molecules. MEK1/2 is activated by a wide variety of growth factors and cytokines, as well as by membrane depolarization and calcium influx (20-23). MEK activates p44 and p42 MAP kinase by phosphorylating both threonine and tyrosine residues at sites located within the activation loop of kinase subdomain VIII. The MEK1/2 (D1A5) Rabbit mAb can be used effectively as a cytoplasmic loading control in cell-based research assays.

1. Budihardjo, I. et al. (1999) Annu Rev Cell Dev Biol 15, 269-90.
2. Schägger, H. (2002) Biochim Biophys Acta 1555, 154-9.
3. Li, P. et al. (1997) Cell 91, 479-89.
4. Liu, X. et al. (1996) Cell 86, 147-57.
5. Du, C. et al. (2000) Cell 102, 33-42.
6. Verhagen, A.M. et al. (2000) Cell 102, 43-53.
7. Srinivasula, S.M. et al. (2001) Nature 410, 112-6.
8. Srinivasula, S.M. et al. (2000) J Biol Chem 275, 36152-7.
9. Gray, C.W. et al. (2000) Eur J Biochem 267, 5699-710.
10. Suzuki, Y. et al. (2001) Mol Cell 8, 613-21.
11. Hegde, R. et al. (2002) J Biol Chem 277, 432-8.
12. Martins, L.M. et al. (2002) J Biol Chem 277, 439-44.
13. van Loo, G. et al. (2002) Cell Death Differ 9, 20-6.
14. Verhagen, A.M. et al. (2002) J Biol Chem 277, 445-54.
15. Fernandes-Alnemri, T. et al. (1994) J Biol Chem 269, 30761-4.
16. Nicholson, D.W. et al. (1995) Nature 376, 37-43.
17. Ostermeier, C. et al. (1996) Curr Opin Struct Biol 6, 460-6.
18. Capaldi, R.A. et al. (1983) Biochim Biophys Acta 726, 135-48.
19. Kadenbach, B. et al. (2000) Free Radic Biol Med 29, 211-21.
20. Crews, C.M. et al. (1992) Science 258, 478-80.
21. Alessi, D.R. et al. (1994) EMBO J 13, 1610-9.
22. Rosen, L.B. et al. (1994) Neuron 12, 1207-21.
23. Cowley, S. et al. (1994) Cell 77, 841-52.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

11940:

IHC / Paraffin, Western Blotting

11967:

IHC / Paraffin, Immunofluorescence*, Immunoprecipitation, Western Blotting

2954:

IHC / Paraffin, Immunofluorescence, Immunoprecipitation, Western Blotting

8727:

Flow, Immunofluorescence, Western Blotting

9665:

Immunoprecipitation, Western Blotting

9745:

Western Blotting

* Product-specific protocol.

Companion Products

• 9038 Cell Fractionation Kit
• 9661 Cleaved Caspase-3 (Asp175) Antibody
• 9662 Caspase-3 Antibody
• 9665 Caspase-3 (8G10) Rabbit mAb
• 5625 Cleaved PARP (Asp214) (D64E10) XP^® Rabbit mAb
• 9542 PARP Antibody
• 9508 Caspase-9 (C9) Mouse mAb
• 7071 Phototope^®-HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 7072 Phototope^®-HRP Western Blot Detection System, Anti-mouse IgG, HRP-linked Antibody
• 7003 20X LumiGLO^® Reagent and 20X Peroxide
• 9998 BSA
• 9999 Nonfat Dry Milk
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 9997 Tris Buffered Saline with Tween 20 (TBST-10X)
• 7722 Blue Loading Buffer Pack
• 7723 Red Loading Buffer Pack
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7076 Anti-mouse IgG, HRP-linked Antibody
• 7075 Anti-biotin, HRP-linked Antibody

These antibodies are available exclusively from Cell Signaling Technology (CST) and are not available from other vendors. 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 7,429,487) from Epitomics, Inc.

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For Research Use Only. Not For Use In Diagnostic Procedures.