Upstream / Downstream

Explore pathways related to this product.

Antibody Guarantee

CST Antibody Performance Guarantee

LEARN MORE  

Questions?

Find answers on our FAQs page.

ANSWERS  

Visit PhosphoSitePlus®

PTM information and tools available.

LEARN MORE

REACTIVITY
H M
Product Includes Volume Solution Color
SAPK/JNK Mouse mAb Coated Microwells 96 tests
Phospho-SAPK/JNK (Thr183/Tyr185) Rabbit Detection Antibody 5.5 ml Green
Anti-rabbit 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

Order Details

Custom Ordering Details: Product is assembled upon order to ensure maximum activity. United States: Please allow up to two weeks for your order to be processed and shipped. Outside of the United States: Please allow up to three weeks, depending on the country, for your order to be processed and shipped. When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.
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® Phospho-SAPK/JNK (Thr183/Tyr185) Chemiluminescent Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-SAPK/JNK (Thr183/Tyr185) 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 SAPK/JNK mouse mAb has been coated on the microwells. After incubation with cell lysates, total SAPK/JNK protein (non-phosphorylated and phosphorylated) is captured by the coated antibody. Following extensive washing, phospho-SAPK/JNK (Thr183/Tyr185) antibody is added to detect the captured phospho-SAPK/JNK (Thr183/Tyr185) protein. Anti-rabbit 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 phospho-SAPK/JNK (Thr183/Tyr185) protein.

Antibodies in kit are custom formulations specific to kit.


Specificity / Sensitivity

PathScan® Phospho-SAPK/JNK (Thr183/Tyr185) Chemiluminescent Sandwich ELISA Kit #7849 detects endogenous levels of phospho-SAPK/JNK (Thr183/Tyr185) in human and mouse 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

The stress-activated protein kinase/Jun-amino-terminal kinase SAPK/JNK is potently and preferentially activated by a variety of environmental stresses including UV and gamma radiation, ceramides, inflammatory cytokines, and in some instances, growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1-MEKK4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4/7. Upon activation, MKKs phosphorylate and activate the SAPK/JNK kinase (2). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (3). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (3). Alternatively, MKK4/7 can be activated in a GTPase-independent mechanism via stimulation of a germinal center kinase (GCK) family member (4). There are three SAPK/JNK genes each of which undergoes alternative splicing, resulting in numerous isoforms (3). SAPK/JNK, when active as a dimer, can translocate to the nucleus and regulate transcription through its effects on c-Jun, ATF-2, and other transcription factors (3,5).


1.  Ichijo, H. (1999) Oncogene 18, 6087-93.

2.  Kyriakis, J.M. and Avruch, J. (2001) Physiol Rev 81, 807-69.

3.  Kyriakis, J.M. (1999) J Biol Chem 274, 5259-62.

4.  Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem. Sci. 23, 481-485.

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

6.  Davis, R.J. (1999) Biochem Soc Symp 64, 1-12.


Entrez-Gene Id 5599
Swiss-Prot Acc. P45983


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.