Product Pathways - Apoptosis / Autophagy

Apoptosis Sampler Kit #9915

• pathway
• more info
• application references
• datasheet PDF
• MSDS PDF
• protocols

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IHC-F=Immunohistochemistry (Frozen)  IF-IC=Immunofluorescence (Immunocytochemistry)  IC=Immunocytochemistry  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  B=Bovine

Specificity / Sensitivity

Each antibody in the Apoptosis Sampler Kit detects endogenous levels of its respective protein. Cleaved Caspase-3 (Asp175), Cleaved Caspase-7 (Asp198) and Cleaved PARP (Asp214) Antibodies recognize only the large fragments of their respective cleaved proteins. Cleaved Caspase-9 (Asp330) Antibody recognizes either the 37 kDa (with prodomain) or 17 kDa large fragment of the cleaved protein. Caspase-3, Caspase-7, Caspase-9 and PARP Antibodies recognize both the full length and large cleaved fragments of their respective proteins.

Western Blotting

Western blot analysis of extracts from Jurkat cells and 293 cells treated with 0.25 mg/ml cytochrome c for 1 hour (cytc) or with 25 µM etoposide for 5 hours (eto), using Cleaved Caspase-7 (Asp198) Antibody #9491.

Western Blotting

Western blot analysis of extracts from Jurkat cells, untreated or etoposide-treated (25 µM), and HeLa cells, untreated or staurosporine-treated (1 µM), using Caspase-7 Antibody #9492.

Western Blotting

Western blot analysis of extracts from HeLa cells, untreated or staurosporine-treated (1 µM), and Jurkat cells, untreated or etoposide-treated, using Cleaved Caspase-9 (Asp330) Antibody (Human Specific) #9501 (upper) or full length Caspase-9 Antibody #9502 (lower).

Western Blotting

Western blot analysis of extracts from HeLa cells, untreated, staurosporine-treated (1 µM) or cytochrome c-treated (0.25 mg/ml), and Jurkat cells, untreated or cytochrome c-treated, using Caspase-9 Antibody (Human Specific) #9502.

Western Blotting

Western blot analysis of extracts from Jurkat cells, untreated or etoposide-treated (25 µM), using Cleaved PARP (Asp214) Antibody (Human Specific) #9541.

Western Blotting

Western blot analysis of extracts from NIH/3T3 cells, untreated or staurosporine-treated (1 µM), and Jurkat cells, untreated or etoposide-treated (25 µM), using PARP Antibody #9542.

Western Blotting

Western blot analysis of extracts from HeLa, NIH/3T3 and C6 cells treated with 1 µM staurosporine in vivo or 0.25 mg/ml cytochrome c in vitro, using Caspase-3 Antibody #9662 (upper) or Cleaved Caspase-3 (Asp175) Antibody #9661 (lower).

Western Blotting

Western blot analysis of extracts from Jurkat, L929 and C6 cells, untreated or treated with 0.25 mg/ml cytochrome c, using Caspase-3 Antibody #9662.

Western Blotting

Western blot analysis of extracts from Jurkat cells, untreated or treated with cytochrome c, showing full length and cleaved caspase-3 and cleaved caspase-3 (19 and 17 kDa) fragments, using Caspase-3 Antibody #9662.

IHC-P (paraffin)

Immunohistochemical staining of paraffin-embedded human tonsil, showing nuclear staining, using PARP Antibody #9542.

IHC-P (paraffin)

Immunohistochemical staining of paraffin-embedded human tonsil, showing cytoplasmic and perinuclear localization of cleaved caspase-3 in apoptotic cells (low and high magnification), using Cleaved Caspase-3 Antibody #9661.

IHC-P (paraffin)

Immunohistochemical staining of paraffin-embedded human tonsil, showing cytoplasmic localization of caspase-3, using Caspase-3 Antibody #9662.

IHC-FL (floating)

Immunostaining of cerebellar neuron culture from newborn rat, using Cleaved Caspase-7 (Asp198) Antibody #9491 (green) and propidium iodide (red). Cultures were treated with 40 µM of tert-Butyl-hydroperoxide (tBH, a free radical) for 30 minutes, then allowed to recover for 3 hours. tBH treatment caused neuronal apoptosis as demonstrated by the condensed nuclei (red). Cleaved caspase-7 is colocalized with apoptotic neurons (green).

IHC-FL (floating)

Confocal micrograph of newborn rat brain tissue, showing control and transient cerebral ischemia, using Cleaved Caspase-3 (Asp175) Antibody #9661 (green) and propidium iodide (red). (Provided by Dr. Bingren Hu, University of Miami School of Medicine, Florida.)

IHC-FL (floating)

Confocal micrograph of newborn rat brain cortex (Cx) and striatum (Str), using Cleaved Caspase-3 (Asp175) Antibody #9661 (green) and propidium iodide (red). (Provided by Dr. Bingren Hu, University of Miami School of Medicine, Florida.)

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-7 (Asp198) Antibody #9491 compared to a nonspecific negative control antibody (red).

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide treated (green), using Caspase-7 Antibody #9492 compared to a nonspecific negative control antibody (red).

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide treated (green), using Cleaved Caspase-9 (Asp330) Antibody (Human Specific) #9501 compared to a nonspecific negative control antibody (red).

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Caspase-9 Antibody (Human Specific) #9502 compared to a nonspecific negative control antibody (red).

Flow Cytometry

Flow cytometric analysis of Pam212 cells, untreated or sulindac sulfone-treated (to induce apoptosis), using Cleaved Caspase-3 (Asp175) Antibody #9661. Results were similar to those obtained by analyzing DNA content; however, the DNA assay can also reflect cell necrosis, not specifically apoptosis. (Provided by Dan Rosson, Ph.D., Lankenau Institute of Medical Research, Wynnewood, PA.)

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide treated (green), using Cleaved Caspase-3 (Asp175) Antibody #9661 compared to a nonspecific negative control antibody (red).

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with synthetic peptides (KLH coupled) corresponding to residues surrounding the proteolytic cleavage sites of human caspase-3, -7 and -9 and human PARP. Antibodies are purified by protein A and peptide affinity chromatography.

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, alpha-fodrin, DFF and lamin A, and induce apoptosis. Cytochrome c released from mitochondria is coupled to the activation of caspase-9, a key initiator caspase (1). Proapoptotic stimuli include the FasL, TNF-alpha, DNA damage and ER stress. Fas and TNFR activate caspases 8 and 10 (2), DNA damage leads to the activation of caspase-9 and ER stress leads to the calcium-mediated activation of caspase-12 (3). The inhibitor of apoptosis protein (IAP) family includes XIAP and survivin and functions by binding and inhibiting several caspases (4,5). Smac/Diablo, a mitochondrial protein, is released into the cytosol upon mitochondrial stress and competes with caspases for binding of IAPs. The interaction of Smac/Diablo with IAPs relieves the inhibitory effects of the IAPs on caspases (6).

1. Baker, S.J. and Reddy, E.P. (1998) Oncogene 17, 3261-3270.
2. Budihardjo, I. et al. (1999) Annu. Rev. Cell Dev. Biol. 15, 269-290.
3. Nakagawa, T. et al. (2000) Nature 403, 98-103.
4. Deveraux, Q. L. et al. (1998) EMBO J. 17, 2215-2223.
5. Li, F. et al. (1998) Nature 396, 580-584.
6. Du, C. et al. (2000) Cell 102, 33-42.

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!

Companion Products

• 9504 Caspase-9 Antibody (Mouse Specific)
• 9505 Cleaved Caspase-9 (Asp315) Antibody (Human Specific)
• 9507 Cleaved Caspase-9 (Asp353) Antibody (Rat Specific)
• 9544 Cleaved PARP (Asp214) Antibody (Mouse Specific)
• 9545 Cleaved PARP (Asp214) Antibody (Rat Specific)
• 9546 Cleaved PARP (Asp214) (19F4) Mouse mAb (Human Specific)
• 9850 Caspase-7 Activity Assay Kit
• 9852 Chaps Cell Extract Buffer (10X)
• 9803 Cell Lysis Buffer (10X)
• 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