Apoptosis array

RayBio® Apoptosis Array



Introduction

Apoptosis is the process of programmed cell death that involves a series of biochemical events leading to a characteristic cell morphology and death. In addition to its importance in biological phenomena such as cell termination, homeostasis, development and lymphocyte interactions, deregulation of apoptosis has been implicated in many diseases such as CAD, HIV infection and cancer. Apoptosis is mediated by a diverse range of extracellular or intracellular cell signals. Following TNF and Fas activation a balance between pro-apoptotic (BAX, BID, BAK, or BAD) and anti-apoptotic (Bcl-Xl and Bcl-2) members of the Bcl-2 family is established. The pro-apoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC from Mitochondria. p53 also plays critical role in apoptosis. Any disruption to the regulation of the p53 will result in impaired apoptosis. Recent reports have also shown the involvement of IGFBP in apoptosis. With RayBio® Human Apoptosis Antibody Array kit, researchers can now simultaneously detect the relative level of 43 apoptosis related proteins in cell lysates. With RayBio® Human Apoptosis antibody Array kit, each of the capture antibodies is printed on membranes or glass slide, and, treated or untreated cell lysate is added onto the array. After extensive washing, a cocktail of biotin-conjugated anti-apoptotic antibody mix is used. After incubation with HRP-Streptavidin, the signals are visualized by chemiluminescene.


Product Highlights

- Relative level of 43 apoptosis –related markers are detected simultaneously
- No need to perform individual immunoprecipitations or Western Blot
- Reliable results with wide range of cell lines
- High specificity, sensitivity and reproducibility
- No special equipment required
- Many choices: chemiluminescence or fluorescence detection system; Membrane or glass chip

How it works




Contents of kit

RayBio® Human Apoptosis Antibody Array kit membranes (2, 4, or 8 membranes)
2X Cell Lysis Buffer (5 ml)
Protease Inhibitor Cocktail (1 tube for 2-4 membranes, and 2 for 8 membranes)
Blocking Buffer (25 ml for less 4 membranes and 50 ml for 8 membranes)
20X Wash Buffer I (30 ml)
20X Wash Buffer II (30 ml)
Cocktail of Biotin-Conjugated Antibody Mix (1 tube for 2 membranes, 2 for 4 membranes, and 4 for 8 membranes)
1,000X HRP-Conjugated Streptavidin (18 µl)
Detection Buffer C (1.5 ml for 2~4 membranes, 2.5 ml for membranes)
Detection Buffer D (1.5 ml for 2~4 membranes, 2.5 ml for 8 membranes)
Eight-Well Tray (1 each)


Representative results



Fig 1. Apoptotic protein profiling in HeLa cell lines 400 ug of cell lysate from human HeLa cell line was incubated overnight with RayBio Human Apoptosis Antibody Array membrane. Control membrane was incubated with blocking buffer. The antibody array membranes were then washed and cocktail of biotinylated antibody mix was used to detect apoptosis-related proteins. After incubation with HRP-Conjugated Streptavidin, the signals were visualized after 5 minute exposure by chemiluminescene.


Proteins detected:

Bad, Bax, Bcl-2, Bcl-w, BID, BIM, Caspase 3, Caspase 8, CD40, CD40L, cIAP-2, cytochrome C, DR6, Fas, Fas ligand, HSP2, HSP70, HTRA, IGF-1, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, IGF-1 sR, Livin, p21, p27, p53, SMAC, Survivin, sTNF-R1, sTNF-R2, TNF-alpha, TNF-beta, TRAIL R1, TRAIL R2, TRAIL R3, TRAIL R4, XIAP

Reference List

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2. Huang RP, Huang R, Fan Y, and Lin Y (2001). A novel method for high- throughput protein profiling from conditioned media and patient’s sera. Ana. Biochem. 294(1):55-62.

3. Lue JI, and George DL (2007). Hepatic IGFBP1 is a prosurvival factor that binds to BAK, protects the liver from apoptosis, and antagonizes the proapoptotic actions of p53 at mitochondria. Genes & Dev. 21: 3095-3109.

4. Thompson, CB (1995). Apoptosis in the pathogenesis and treatment of disease. Science. 267 (5203): 1456–62.

5. Brüne B (2003). Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ. 10 (8): 864–9.

6. Fesik SW, Shi Y (2001). Controlling the caspases. Science. 294 (5546): 1477–8.

7. Dejean LM, Martinez-Caballero S, Manon S, and Kinnally KW (2006). Regulation of the mitochondrial apoptosis-induced channel, MAC, by BCL-2 family proteins. Biochim. Biophys. Acta. 1762 (2): 191–201.

8. Wajant H (2002). The Fas signaling pathway: more than a paradigm. Science. 296 (5573): 1635–6.

9. Chen G, and Goeddel DV (2002). TNF-R1 signaling: a beautiful pathway. Science. 296 (5573): 1634–5.

10. Murphy KM, Ranganathan V, Farnsworth ML, Kavallaris M, and Lock RB ( 2000). Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. Cell Death Differ. 7 (1): 102–11.

11. Susin SA, Lorenzo HK, and Zamzami N (1999). Molecular characterization of mitochondrial apoptosis-inducing factor. Nature. 397 (6718): 441–6.

12. Nagata S (2000). Apoptotic DNA fragmentation. Exp. Cell Res. 256 (1): 12–8.