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Figure 1. Target map of the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856.

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Figure 2. HT-29 cells were grown to 80% confluency and then either untreated (left panel) or UV-irradiated and allowed to recover for 60 minutes (right panel). Cell extracts were prepared and analyzed using the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856. Images were acquired by briefly exposing the slide to standard chemiluminescent film.

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Figure 3. HeLa cells were grown to 90% confluency and then either untreated (left panel) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (100 ng/ml, 20 min; right panel). Cell extracts were prepared and analyzed using the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856. Images were acquired by briefly exposing the slide to standard chemiluminescent film.

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Figure 4. HeLa cells were grown to 90% confluency and then serum starved overnight. Cells were either untreated (left panel) or treated with Human Transforming Growth Factor β3 (hTGF-β3) #8425 (100 ng/ml, 20 min; right panel). Cell extracts were prepared and analyzed using the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856. Images were acquired by briefly exposing the slide to standard chemiluminescent film.

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Figure 5. HeLa cells were grown to 90% confluency and then either untreated (left panel) or treated with Staurosporine #9953 (1 μM, 3.5 hr; right panel). Cell extracts were prepared and analyzed using the PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) #12856. Images were acquired by briefly exposing the slide to standard chemiluminescent film.

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PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) Protocol

A. Preparing Cell Lysates

  1. Thaw 1X Cell Lysis Buffer #7018 and mix thoroughly. Supplement 1X Cell Lysis Buffer with a Protease Inhibitor Cocktail (100X) #5871. Keep lysis buffer on ice.
  2. Remove media and wash cells once with ice-cold 1X PBS.
  3. Remove PBS and add ice-cold 1X Cell Lysis Buffer. For adherent cells, use 0.5 ml 1X Cell Lysis Buffer #7018 for each plate (10 cm in diameter). Incubate on ice for 2 minutes.
  4. Transfer lysates to a microcentrifuge tube and microcentrifuge at maximum speed for 2 minutes at 4°C.
  5. Transfer the supernatant to a new tube. The supernatant is the cell lysate and may be used immediately or stored at –80°C in single-use aliquots.
  6. Dilute lysates to 0.2 – 1.0 mg/ml in Array Diluent Buffer immediately before performing the assay. Set aside on ice.

B. Assay Procedure

  1. Bring glass slides and blocking buffer to room temperature before use.
  2. Prepare 1X Array Wash Buffer by diluting 20X Array Wash Buffer in deionized water. Dilute 1 ml of 20X Array Wash Buffer with 19 ml of deionized water. Label as 1X Array Wash Buffer. Keep at room temperature.
  3. Prepare 1X Detection Antibody Cocktail as follows:

    For running only 1 slide: Dilute 150 µl of 10X Detection Antibody Cocktail with 1350 µl of Array Diluent Buffer.

    For running 2 slides: Dilute 300 µl of 10X Detection Antibody Cocktail with 2700 µl of Array Diluent Buffer. Keep on ice.

  4. Prepare 1X HRP-linked Streptavidin by diluting 10X HRP-linked Streptavidin in Array Diluent Buffer. Keep on ice.
  5. Affix the multi-well gasket to the glass slide (see figure at bottom):
    1. Place the multi-well gasket facedown on the bench top (the silicone layer should be facing up). Remove the protective plastic film.
    2. Carefully place the glass slide on top of the multi-well gasket with the nitrocellulose pads facing down while aligning the pads with the openings in the gasket. The orientation line should appear in the upper left hand corner when the slide is oriented vertically.
    3. Insert the metal clip into the groove in the gasket and rotate the clip into the locked position. Ensure that the clip is on the same side as the orientation line on the slide.

      NOTE: one of the clips has a small dot etched onto the upper rib to assist with pad designation (see slide assembly photos).

    4. Slide the clip into place.
    5. Snap the unmarked metal clip to the other side of the assembly in the same manner and slide into place.
    6. The assembled array is ready to use.
  6. Add 100 µl Array Blocking Buffer to each well and cover with sealing tape. Incubate for 15 minutes at room temperature on an orbital shaker.

    NOTE: Do not allow the pads to dry out at any time during the assay.

  7. Decant Array Blocking Buffer by gently flicking out the liquid into a sink or other appropriate waste receptacle. Add 50 – 75 µl diluted lysate to each well and cover with sealing tape. Incubate for 2 hours at room temperature (or overnight at 4°C) on an orbital shaker.
  8. Decant well contents by gently flicking out the liquid into a sink or other appropriate waste receptacle. Add 100 µl 1X Array Wash Buffer to each well and incubate for 5 minutes at room temperature on an orbital shaker. Repeat three more times. Decant well contents.
  9. Add 75 µl 1X Detection Antibody Cocktail to each well and cover with sealing tape. Incubate for 1 hour at room temperature on an orbital shaker.
  10. Wash 4 – 5 minutes with 100 µl 1X Array Wash Buffer as in step 8.
  11. Add 75 µl 1X HRP-linked Streptavidin to each well and cover with sealing tape. Incubate for 30 minutes at room temperature on an orbital shaker.
  12. Wash 4 – 5 minutes with 100 µl 1X Array Wash Buffer as in step 8.
  13. Remove multi-well gasket by pulling the bottom of the metal clips away from the center of the slide, then peeling the slide and gasket apart.
  14. Place the slide face up in a plastic dish (a clean pipette tip box cover works well). Wash briefly with 10 ml 1X Array Wash Buffer.
  15. Dilute and combine LumiGLO® and Peroxide reagents immediately before use (to make 10 ml of a 1X solution, combine 9 ml deionized water with 0.5 ml of 20X LumiGLO® and 0.5 ml of 20X Peroxide).

    Note for Kodak® Biomax® film users: This dilution of LumiGLO®/Peroxide may necessitate very short exposure times (2-3 seconds) for some targets. For more convenient exposure times (20‑30 seconds) add 20 ml of deionized water to the 10 ml LumiGLO®/Peroxide mix to make a 3 fold more diluted chemiluminescent reagent.

  16. Decant Array Wash Buffer and cover slide with LumiGLO®/Peroxide reagent.
  17. Transfer slide to sheet protector, ensuring that it is still covered by LumiGLO®/Peroxide reagent (add a small amount on top of the slide).
  18. Immediately capture an image of the slide using a digital imaging system capable of detecting chemiluminescent signals. If desired, quantify spot intensities using commercially available array image analysis software. Alternatively, chemiluminescent film may be used. Expose film for 2 – 30 seconds using even and light pressure on the top of the development cassette (do not fasten the cassette clamps) to avoid squeezing out the LumiGLO®/ Peroxide reagent. Develop the film using an automated film developer.

    NOTE: If both slides are being used, it is not recommended to expose them simultaneously in the same development cassette. In this case, leave the second slide in the wash buffer (step 12) while proceeding with steps 13 – 18 using the first slide. After the first slide is finished, proceed with steps 13 – 18 using the second slide and freshly diluted LumiGLO®/Peroxide reagent.

Slide Assembly Photos

LumiGLO® is a registered trademark of Kirkegaard and Perry Laboratories.

Kodak® and BioMax® are trademarks of Eastman Kodak Company.

posted November 2013

Product Includes Quantity Cap Color
Array Slides - Stress and Apoptosis Array 2 ea
16-Well Gasket 2 ea
Sealing Tape 2 sheets
Chemiluminescent Development Folder
20X Array Wash Buffer 15 ml White
Array Blocking Buffer 5 ml Red
Array Diluent Buffer 15 ml Blue
Detection Ab Cocktail 10X - Stress and Apoptosis Array 300 µl White
HRP-Linked Streptavidin (10X) 300 µl Clear
20X LumiGLO® Reagent and 20X Peroxide 7003 5 ml each Brown
PathScan® Sandwich ELISA Lysis Buffer (1X) 7018 30 ml Clear

Product Usage Information

Storage: Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).

Product Description

The PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 19 signaling molecules that are involved in the regulation of the stress response and apoptosis. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two slides allowing for the interrogation of 32 different samples and the generation of 608 data points in a single experiment. Cell lysates are incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated HRP and LumiGLO® Reagent are then used to visualize the bound detection antibody by chemiluminescence. An image of the slide can be captured with either a digital imaging system or standard chemiluminescent film. The image can be analyzed visually or the spot intensities quantified using array analysis software.


Specificity / Sensitivity

PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout) detects the target proteins as specified on the Array Target Map. No substantial cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see kit protocol).


Species Reactivity: Human

Cell death can occur due to a variety of circumstances including nutrient deprivation, inability to generate or store the energy required for metabolic functions, or deleterious environment that causes irreparable damage. Cells integrate multiple signals from a variety of sources before following either pro- or anti-apoptotic pathways. These signals can often carry conflicting information. Assessing the net effect of these processes in cell populations can be achieved by monitoring changes in a number of key signaling components. The caspase-3 and caspase-7 proteases exert a pro-apoptotic function through cleavage of multiple cellular targets. Caspase-3 and caspase-7 are activated by cleavage at Asp175 and Asp198, respectively. PARP is a DNA repair and apoptosis enzyme that is inactivated by cleavage at Asp214 by caspase-3 or caspase-7. HSP27 is a mediator of cell stress that confers resistance to adverse environmental conditions. HSP27 is activated by phosphorylation at Ser82. Chk1 and Chk2 kinases act downstream of ATM/ATR and play an important role in DNA damage checkpoint control. Activation of Chk1 and Chk2 involve phosphorylation at Ser345 and Thr68, respectively. Tumor suppressor p53 plays an important role in cellular response to DNA damage. p53 is phosphorylated at Ser15 by ATM/ATR or DNA-PK leading to its accumulation. Smad2 is a key mediator of TGF-β signaling. Stimulation by TGF-β leads to Smad2 phosphorylation at Ser465/467 and translocation of Smad2 into the nucleus. The outcome of TGF-β signaling is context dependent and can either induce apoptosis or contribute to tumor cell metastasis. Activation of NF-κB/Rel occurs through a proteasome-mediated degradation of IκBα. The inhibitor IκBα is targeted to the proteasome via phosphorylation of IκBα at Ser32 and Ser36. NF-κB activation is triggered by a diverse group of extracellular signals promoted by inflammatory cytokines, growth factors, and chemokines. TAK1 is a kinase that can be activated by TGF-β, bone morphogenetic proteins and other cytokines. Activated TAK1 phosphorylates MKK4, MKK3/6, and NIK. Phosphorylation of TAK1 at Ser412 is one of the mechanisms that regulate the levels of its activation. Cellular stress such as viral infection, endoplasmic reticulum stress, and amino acid deprivation leads to phosphorylation of eIF2α. Phosphorylation of eIF2α at Ser51 in response to cellular stress leads to a reduction of protein synthesis. The ERK1 and ERK2 MAP kinases are major signaling nodes that have many substrates and primarily transmit growth and proliferation signals. The ERK MAP kinase is activated by a dual phosphorylation of Thr202 and Tyr204. p38 MAPK and SAPK/JNK MAP kinases are activated through a similar dual phosphorylation mechanism in response to pro-inflammatory cytokines and genotoxic stress. Akt is activated by stimulation of growth-factor receptors and primarily promotes anabolic growth and survival signals via targeting its broad array of substrates. Akt phosphorylates Bad at Ser136 and inhibits its ability to induce apoptosis. Survivin is an anti-apoptotic protein that is highly expressed during fetal development and cancer cell malignancy. Survivin binds and inhibits caspase-3, controlling the cell cycle by inhibiting apoptosis and promoting cell division. α-tubulin is a building block of microtubules that are present in all eukaryotic cells. The levels of the globular α-tubulin are considered to remain relatively constant. Therefore, assessing the relative levels of α-tubulin may assist with signal normalization between the various samples.


1.  Cohen, G.M. (1997) Biochem. J. 326, 1-16.

2.  Bratton, S.B. and Cohen, G.M. (2001) Trends Pharmacol Sci 22, 306-15.

3.  Green, D.R. and Reed, J.C. (1998) Science 281, 1309-12.

4.  Basu, S. and Kolesnick, R. (1998) Oncogene 17, 3277-85.

5.  Jeremias, I. and Debatin, K.M. (1998) Eur Cytokine Netw 9, 687-8.

6.  Aravind, L. et al. (1999) Trends Biochem Sci 24, 47-53.

7.  Bates, S. and Vousden, K.H. (1999) Cell Mol Life Sci 55, 28-37.

8.  Heyninck, K. and Beyaert, R. (2001) Mol Cell Biol Res Commun 4, 259-65.

9.  Abe, K. et al. (2000) Ann N Y Acad Sci 926, 52-63.

10.  Rath, P.C. and Aggarwal, B.B. (1999) J Clin Immunol 19, 350-64.

11.  Holoch, P.A. and Griffith, T.S. (2009) Eur J Pharmacol 625, 63-72.

12.  Boatright, K.M. and Salvesen, G.S. (2003) Curr Opin Cell Biol 15, 725-31.

13.  Gjertsen, B.T. and Døskeland, S.O. (1995) Biochim Biophys Acta 1269, 187-99.

14.  Clemens, M.J. (2001) Prog Mol Subcell Biol 27, 57-89.

15.  Janes, K.A. et al. (2005) Science 310, 1646-53.

16.  Janes, K.A. et al. (2008) Cell 135, 343-54.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.

12856
PathScan® Stress and Apoptosis Signaling Antibody Array Kit (Chemiluminescent Readout)