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REACTIVITY
H M R
Product Includes Volume Solution Color
c-Jun mAb coated Microwells 96 tests
c-Jun Detection Ab 5.5 ml Green
Anti-mouse IgG, HRP-linked Antibody 5.5 ml Red
Luminol/Enhancer Solution 3 ml Colorless
Stable Peroxide Buffer 3 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) 9803 15 ml Yellowish
Page

ELISA Chemiluminescent

NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature. This /product/productDetail.jsp?productId=luminescent ELISA is offered in low volume microplate. Samples and reagents only require 50 µl per microwell.

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 1X 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.

  5. 20X LumiGLO® Reagent and 20X Peroxide: (#7003).

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 medium 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 used 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 50 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 room temperature. 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, 150 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 dry completely at any time.
    4. Clean the underside of all wells with a lint-free tissue.
  5. Add 50 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at room temperature for 1 hr.
  6. Repeat wash procedure (Section C, Step 4).
  7. Add 50 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate at room temperature for 30 min.
  8. Repeat wash procedure (Section C, Step 4).
  9. Prepare detection reagent working solution by mixing equal parts 2X LumiGLO® Reagent and 2X Peroxide.
  10. Add 50 µl of the detection reagent working solution to each well.
  11. Use a plate-based luminometer set at 425 nm to measure Relative Light Units (RLU) within 1–10 min following addition of the substrate.
    1. Optimal signal intensity is achieved when read within 10 min.

posted November 2009

revised September 2013

protocol id: 41

Product Description

The PathScan® Total c-Jun Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total c-Jun protein with a chemiluminescent readout. Chemiluminescent ELISAs often have a wider dynamic range and higher sensitivity than conventional chromogenic detection. This chemiluminescent ELISA, which is offered in low volume microplates, shows increased signal and sensitivity while using a smaller sample size. A c-Jun rabbit mAb has been coated on the microwells. After incubation with cell lysates, c-Jun proteins are captured by the coated antibody. Following extensive washing, a c-Jun mouse detection mAb is added to detect the captured c-Jun protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of total c-Jun protein.

Antibodies in kit are custom formulations specific to kit.


Specificity / Sensitivity

PathScan® Total c-Jun Chemiluminescent Sandwich ELISA Kit #7028 detects endogenous levels of total c-Jun in human, mouse and rat cells. 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, Mouse, Rat

c-Jun is a member of the Jun family containing c-Jun, JunB, and JunD, and is a component of the transcription factor activator protein-1 (AP-1). AP-1 is composed of dimers of Fos, Jun, and ATF family members and binds to and activates transcription at TRE/AP-1 elements (reviewed in 1). Extracellular signals including growth factors, chemokines, and stress activate AP-1-dependent transcription. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through SAPK/JNK (reviewed in 2). Knock-out studies in mice have shown that c-Jun is essential for embryogenesis (3), and subsequent studies have demonstrated roles for c-Jun in various tissues and developmental processes including axon regeneration (4), liver regeneration (5), and T cell development (6). AP-1 regulated genes exert diverse biological functions including cell proliferation, differentiation, and apoptosis, as well as transformation, invasion and metastasis, depending on cell type and context (7-9). Other target genes regulate survival, as well as hypoxia and angiogenesis (8,10). Research studies have implicated c-Jun as a promising therapeutic target for cancer, vascular remodeling, acute inflammation, and rheumatoid arthritis (11,12).


1.  Jochum, W. et al. (2001) Oncogene 20, 2401-12.

2.  Davis, R.J. (2000) Cell 103, 239-52.

3.  Hilberg, F. et al. (1993) Nature 365, 179-81.

4.  Raivich, G. et al. (2004) Neuron 43, 57-67.

5.  Behrens, A. et al. (2002) EMBO J 21, 1782-90.

6.  Riera-Sans, L. and Behrens, A. (2007) J Immunol 178, 5690-700.

7.  Leppä, S. and Bohmann, D. (1999) Oncogene 18, 6158-62.

8.  Shaulian, E. and Karin, M. (2002) Nat Cell Biol 4, E131-6.

9.  Weiss, C. and Bohmann, D. (2004) Cell Cycle 3, 111-3.

10.  Karamouzis, M.V. et al. (2007) Mol Cancer Res 5, 109-20.

11.  Kim, S. and Iwao, H. (2003) J Pharmacol Sci 91, 177-81.

12.  Dass, C.R. and Choong, P.F. (2008) Pharmazie 63, 411-4.


Entrez-Gene Id 3725
Swiss-Prot Acc. P05412


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.