RayBiotech,Inc.

Product: RayBio® Apoptosis Array

Introduction

Product Highlights

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

Representative Data#1(Membrane based):

Apoptotic protein profiling in drug-treated Jurkat cultured cells: Jurkat cells were treated with Apoptosis Inducer Set (BioVision, Inc. cat# K121-5) for 7 hours (10mM Actinomycin D, 2mM Camptothecin, 100mM Cycloheximide, 10mM Dexamethasone, and 100mM Etopiside). 500 mg of cell lysates from both induced and uninduced Jurkat cells were incubated overnight with RayBio® Human Apoptosis Antibody Array membranes. Control membrane was incubated with blocking buffer. The membranes were then washed and cocktail of biotinylated antibodies was used to detect bound proteins. After incubation with HRP-streptavidin, the signals were visualized by chemiluminescence.

Apoptotic protein profiling in drug-treated Jurkat cultured cellsArray data is confirmed by Western Analysis

Representative Data#2 (Slide based):

Apoptotic protein profiling in Induced and uninduced Jurkat cell lines: Jurkat cells were treated with apoptosis inducer set for 7 hours (10mM Actinomycin D, 2mM Camptothecin, 100mM Cycloheximide, 10mM Dexamethasone, and 100mM Etopiside). 50 mg of cell lysate from both induced and uninduced Jurkat cells were 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. Array results are shown in Figure A for uninduced Jurkat cells and Figure B for induced Jurkat cells. Representative markers are highlighted in the numbered rectangular boxes. Figure C shows Western Blotting of apoptotic markers (Bcl-2. Caspase-3, HTRA-2) using both induced (0 Hr) and induced (7 Hr) Jurkat cell lysates.

Price

Membrane-Based Arrays:

Product	Cat#	Size	Detection	Species	Sample type	Price
RayBio® Human Apoptosis Antibody Array (2)	AAH-APO-1-2	2 membranes for 2 samples	Chemiluminescence	Human	Cell lysates	$298.00
RayBio® Human Apoptosis Antibody Array (4)	AAH-APO-1-4	4 membranes for 4 samples	Chemiluminescence	Human	Cell lysates	$489.00
RayBio® Human Apoptosis Antibody Array (8)	AAH-APO-1-8	8 membranes for 8 samples	Chemiluminescence	Human	Cell lysates	$880.00
RayBio® Human Apoptosis Antibody Array Service	AAH-APO-SV	Minumum 2 samples	Chemiluminescence	Human	Cell lysates	Inquiry

Glass Chip-Based Arrays:

Product	Cat#	Size	Detection	Species	Sample type	Price
RayBio® Human Apoptosis Antibody Array (4)	AAH-APO-G1-4	1slide for 4 samples	Fluorescence	Human	Cell lysates	$428.00
RayBio® Human Apoptosis Antibody Array (8)	AAH-APO-G1-8	2 slides for 8 samples	Fluorescence	Human	Cell lysates	$728.00
RayBio® Human Apoptosis Antibody Array Service	AAH-APO-GSV	Minumum 4 samples	Fluorescence	Human	Cell lysates	Inquiry

Map

Quantibody Arrays - quantitative multiplex ELISA arrays to measure protein concentrations

C-Series Arrays - membrane based semi-quantitative array requires no specialized equipment

G-Series arrays - glass slide based semi-quantitative array uses as little as 10 ul of your sample

Label based antibody arrays - highest density array to detect up to 507 proteins in one sample

ELISA kits - over 200 ELISA kits to choose from

Cell-based Phosphorylation ELISA - measures relative levels of phosphorylation without sample prep

Antibodies - over 10,000 recombinant, monoclonal, and polyclonal antibodies

Proteins - large collection of proteins

Peptides - thousands of peptides available

Enzyme Immunoassay (EIA) - competitive immunoassay to measure peptide and protein concentrations

Testing Services - you send us your samples; we send you results

Apoptosis Array - detect 43 apoptotic proteins in one sample

Custom Services - recombinant, monoclonal, polyclonal antibodies ; recombinant protein; peptide synthesis

Phosphorylation Antibody Arrays - detect phosphorylation of multiple proteins simultaneously

Reference List

1. Zaparta JM, Krajewska M, Krajewski S, Huang RP, Takayama S, Wang HG, Adamson E, and Reed JR (1998). Expression of multiple apoptosis-regualtory genes in human breast cancer cell lines and primary tumors. Breast Can Res &Treat. 47: 129-140.

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.

SuperIT