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7105
PathScan® Apoptosis Multi-Target Sandwich ELISA Kit

PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105

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Figure 1. Treatment of HeLa cells with doxorubicin induces phosphorylation of p53 at Ser15, as well as cleavage of PARP and caspase-3 as detected by PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 and Western analysis. HeLa cells (80-90% confluent) were starved overnight and stimulated with doxorubicin (5 μM at 37ºC for indicated times). Lysates were assayed at a protein concentration of 1 mg/ml. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left panel, while the corresponding Western blots are shown in the right panel. Antibodies used for Western analysis include Phospho-p53 (Ser15) Antibody #9284, p53 Antibody #9282, Cleaved Caspase-3 (Asp175) Antibody #9661 and PARP Antibody #9542. Total Bad and phospho-Bad (Ser112) proteins were not detected by Western due to low endogenous levels in HeLa cells.

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Figure 2. Treatment of HeLa cells with staurosporine induces cleavage of PARP and caspase-3 but not phosphorylation of p53 at Ser15 as detected by PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 and Western analysis. HeLa cells (80-90% confluent) were starved overnight and stimulated with staurosporine (2 μM at 37ºC for indicated times). Lysates were assayed at a protein concentration of 1 mg/ml. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left panel, while the corresponding Western blots are shown in the right panel. The antibodies used for the Western analyses include Phospho-p53 (Ser15) Antibody #9284, p53 Antibody #9282, Cleaved Caspase-3 (Asp175) Antibody #9661 and PARP Antibody #9542. Total Bad and Phospho-Bad (Ser112) proteins were not detected by Western due to low endogenous levels in HeLa cells.

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Figure 3. Treatment of COS cells with doxorubicin induces low levels apoptosis as detected by PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 and Western analysis. COS cells (80-90% confluent) were starved overnight and stimulated with doxorubicin (5 μM for at 37ºC for indicated times). Lysates were assayed at a protein concentration of 1 mg/ml. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left panel, while the corresponding Western blots are shown in the right panel. The antibodies used for the Western analyses include Phospho-p53 (Ser15) Antibody #9284, p53 Antibody #9282, Cleaved Caspase-3 (Asp175) Antibody #9661, PARP Antibody #9542, Phospho-Bad (Ser112) (7E11) Mouse mAb #9296 and Bad Antibody #9292.

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Schematic Image 4

Figure 4. Schematic representation of a 96-well plate depicting the color-code of the reagents used to detect endogenous levels of Phospho-p53 (Ser15) (orange; 1 & 2), p53 (yellow; 3 & 4), Cleaved Caspase 3 (Asp175) (purple; 5 & 6), Cleaved PARP (Asp214) (green; 7 & 8), Phospho-Bad (Ser112) (pink; 9 & 10) and Bad protein (grey; 11 & 12).

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Product Includes Volume Solution Color
Apoptosis Multi-Target 96 tests
p53 Rabbit mAb Coated Microwells 16 tests
Phospho-p53 (S15) Mouse Detection Antibody 1.8 ml Green
Anti-mouse IgG, HRP-linked Antibody 1.8 ml Red
p53 Rabbit mAb Coated Microwells 16 tests
p53 Mouse Detection Antibody 1.8 ml Green
Anti-mouse IgG, HRP-linked Antibody 1.8 ml Red
Cleaved-Caspase 3 (D175) Rabbit mAb coated Microwells 16 tests
Biotinylated Caspase-3 Rabbit Detection Antibody 1.8 ml Green
HRP-linked Streptavidin 1.8 ml Red
PARP (D214) Rabbit Antibody Coated Microwells 16 tests
Biotinylated PARP Rabbit Detection Antibody 1.8 ml Green
HRP-linked Streptavidin 1.8 ml Red
Bad Rabbit Antibody Coated Microwells 16 tests
Phospho-Bad (S112) Mouse Detection Antibody 1.8 ml Green
Anti-mouse IgG, HRP-linked Antibody 1.8 ml Red
Bad Rabbit Antibody Coated Microwells 16 tests
Bad Mouse Detection Antibody 1.8 ml Green
Anti-mouse IgG, HRP-linked Antibody 1.8 ml Red
TMB Substrate 7004 11 ml Colorless
STOP Solution 7002 11 ml Colorless
Sealing Tape 2 ea
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) 9803 15 ml Yellowish

ELISA Colorimetric

NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L PBS: add 50 ml 10X PBS to 950 ml dH2O, mix.
  2. Bring all microwell strips to room temperature before use.
  3. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in dH2O.
  4. 1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).

    Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.

    NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.

  5. TMB Substrate: (#7004).
  6. STOP Solution: (#7002).

B. Preparing Cell Lysates

For adherent cells

  1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
  4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  5. Sonicate lysates on ice.
  6. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
  3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

C. Test Procedure

  1. After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed in the storage bag and stored at 4°C immediately.
  2. Cell lysates can be undiluted or diluted with sample diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results.
  3. Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C.
  4. Gently remove the tape and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash 4 times with 1X wash buffer, 200 µl each time per well.
    3. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
    4. Clean the underside of all wells with a lint-free tissue.
  5. Add 100 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at 37°C for 1 hr.
  6. Repeat wash procedure (Section C, Step 4).
  7. Add 100 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate for 30 min at 37°C.
  8. Repeat wash procedure (Section C, Step 4).
  9. Add 100 µl of TMB substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C.
  10. Add 100 µl of STOP solution to each well. Shake gently for a few seconds.

    NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.

  11. Read results
    1. Visual Determination: Read within 30 min after adding STOP solution.
    2. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP solution.

posted June 2005

revised November 2013

CST’s PathScan® Apoptosis Multi-Target Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of p53 protein, phospho-p53 protein (Ser15), Bad, phospho-Bad (Ser112), Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214). These molecules represent key signaling proteins in pathways controlling survival and apoptosis. Sixteen assays are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual sandwich ELISA kits*. Briefly, a capture antibody** has been coated onto the microwells. After incubation with cell lysates, the target protein is captured by the coated antibody. Following extensive washing, a detection antibody** is added to detect the captured target protein. An HRP-linked secondary antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of bound target protein.

*See companion products.

**Antibodies in kit are custom formulations specific to kit.

CST's PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 detects endogenous levels of six proteins: total p53, phospho-p53 (Ser15), total Bad, phospho-Bad (Ser112), cleaved caspase-3 (Asp175) and cleaved PARP (Asp214). Activation of these proteins can be observed over time in response to toxic chemical compounds. As shown in Figures 1 and 2, both doxorubicin and staurosporine can induce apoptosis in HeLa cells, evidenced by increased levels of cleaved PARP and caspase-3. However, treatment with doxorubicin, which damages cellular DNA, induces p53 phosphorylation at Ser15 and stabilizes p53, while treatment with staurosporine, a kinase inhibitor, has no effect on p53 phosphorylation. While total Bad and phospho-Bad (Ser112) levels are relatively consistent after doxorubicin treatment, a gradual decline of both targets was observed after staurosporine treatment. COS cells are resistant to apoptosis due to high constitutive levels of p53. Therefore, the same dose of doxorubicin applied to HeLa cells only induces low amounts of apoptosis in these cells as evidenced by cleaved caspase-3 and cleaved PARP protein levels (Figure 3). The relationship between the protein concentration of the lysate and the absorbance at 450 nm can be found in the datasheets associated with the individual PathScan® Sandwich ELISA Kits*.

*See companion products.

This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

Human, Monkey

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. Cytochrome c released from mitochondria is coupled to the activation of caspase-9, a key initiator caspase (1). Proapoptotic stimuli include the FasL, TNF-α, 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, 269-90.
  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.
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.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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