JNK 1/2 (pT183/Y185 + Total) ELISA Kit (ab176662)

Overview

  • Product name
    JNK 1/2 (pT183/Y185 + Total) ELISA Kit
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    (pT183/Y185) 6 2.7%
    (Total) 6 2.8%
    Inter-assay
    Sample n Mean SD CV%
    (pT183/Y185) 3 3.8%
    (Total) 3 3.3%
  • Sample type
    Cell Lysate, Tissue Homogenate
  • Assay type
    Semi-quantitative
  • Assay time
    1h 30m
  • Assay duration
    One step assay
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Product overview

    Abcam’s JNK1/2 (pT183/Y185) and JNK1/2 (Total) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of JNK1/2 (pT183/Y185) and Total JNK1/2 protein in Human and mouse cells.

    The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • Notes

    Estimated sensitivity: Phospho-JNK1/2 (Thr183/Tyr185): 0.5 ng/mL (tested with recombinant protein), Total JNK1/2: 0.1 ng/mL (tested with recombinant protein)
    Range: Phospho-JNK1/2 (Thr183/Tyr185): 1-100 ng/mL, Total JNK1/2: 0.2-20 ng/mL

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Wash Buffer PT 1 x 15ml
    50X Cell Extraction Enhancer Solution 1 x 1ml
    5X Cell Extraction Buffer PTR (ab193970) 1 x 12ml
    JNK1/2 (pT183/Y185) Capture Antibody 1 x 1.5ml
    JNK1/2 (pT183/Y185) Detector Antibody 1 x 1.5ml
    JNK1/2 (Total) Capture Antibody 1 x 1.5ml
    JNK1/2 (Total) Detector Antibody 1 x 1.5ml
    Lyophilized JNK1/2 Control Lysate 1 vial
    Plate Seal 1 unit
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Substrate 1 x 12ml
  • Research areas
  • Function
    Responds to activation by environmental stress and pro-inflammatory cytokines by phosphorylating a number of transcription factors, primarily components of AP-1 such as JUN, JDP2 and ATF2 and thus regulates AP-1 transcriptional activity. In T-cells, JNK1 and JNK2 are required for polarized differentiation of T-helper cells into Th1 cells (By similarity). Phosphorylates heat shock factor protein 4 (HSF4).
    JNK1 isoforms display different binding patterns: beta-1 preferentially binds to c-Jun, whereas alpha-1, alpha-2, and beta-2 have a similar low level of binding to both c-Jun or ATF2. However, there is no correlation between binding and phosphorylation, which is achieved at about the same efficiency by all isoforms.
  • Sequence similarities
    Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. MAP kinase subfamily.
    Contains 1 protein kinase domain.
  • Domain
    The TXY motif contains the threonine and tyrosine residues whose phosphorylation activates the MAP kinases.
  • Post-translational
    modifications
    Dually phosphorylated on Thr-183 and Tyr-185, which activates the enzyme.
  • Information by UniProt
  • Alternative names
    • c Jun kinase 2
    • c Jun N terminal kinase 2
    • c Jun N terminal kinase 3
    • c-Jun N-terminal kinase 1
    • JNK 55
    • JNK-46
    • JNK1
    • JNK1A2
    • JNK2
    • JNK21B1/2
    • JNK2A
    • JNK2ALPHA
    • JNK2B
    • JNK2BETA
    • JNK3
    • JNK3 alpha protein kinase
    • JNK3A
    • Jun kinase
    • JUN N terminal kinase
    • MAP kinase 10
    • MAP kinase 8
    • MAP kinase 9
    • MAP kinase p49 3F12
    • MAPK 10
    • MAPK 8
    • MAPK 9
    • MAPK10
    • mapk8
    • MAPK9
    • Mitogen activated protein kinase 10
    • Mitogen activated protein kinase 8 isoform JNK1 alpha1
    • Mitogen activated protein kinase 8 isoform JNK1 beta2
    • Mitogen activated protein kinase 9
    • Mitogen-activated protein kinase 8
    • MK08_HUMAN
    • p493F12
    • p54a
    • p54aSAPK
    • p54bSAPK
    • PRKM10
    • PRKM8
    • PRKM9
    • SAPK
    • SAPK1
    • SAPK1a
    • SAPK1b
    • SAPK1c
    • Stress activated protein kinase 1a
    • Stress activated protein kinase 1b
    • Stress activated protein kinase 1c
    • Stress activated protein kinase beta
    • Stress activated protein kinase JNK2
    • Stress activated protein kinase JNK3
    • Stress-activated protein kinase 1
    • Stress-activated protein kinase JNK1
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab176662 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Example of a typical JNK1/2 (pT183/Y185) and JNK1/2 (Total) cell lysate dilution series. Background-subtracted data values (mean +/- SD) are graphed.

  • Example of a typical JNK1/2 (pT183/Y185) recombinant protein standard curve. The proportion of total protein that is phosphorylated is unknown - data is indicative only. Background-subtracted data values (mean +/- SD) are graphed.

  • Linearity of dilution in representative sample matrices. Cellular lysates were prepared at 3 concentrations in common media containing 1X Cell Extraction Buffer PTR. Data from duplicate measurements of JNK1/2 (pT183/Y185) are normalized and plotted.

  • Cell line analysis for Total JNK1/2 from 200 µg/mL preparations of cell extracts. Data from triplicate measurements (mean +/- SD) are plotted and compared to 1X Cell Extraction Buffer PTR (zero).

  • Induction of JNK1/2 (pT183/Y185) phosphorylation in HeLa cells in response to anisomycin treatment. HeLa cells were cultured in 96-well tissue culture plates and treated (30 min) with a dose-range of anisomycin before cell lysis. Data from quadruplicate measurements of JNK1/2 (pT183/Y185) are plotted and compared against Total JNK1/2 protein levels. Comparative JNK1/2 (pT183/Y185) and JNK1/2 (Total) data also shown by Western Blot.

Protocols

References

ab176662 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Abreviews
We are actually working on the preservation of rat pancreas from ischemia-reperfusion injury that occurs during the retrieval of the organ before a transplantation to a recipient. We are looking for strong ischemia markers and we know from literature that JNK activation contributes to myocardial IR injury after prolonged hypothermic storage (Vassalli G et al., 2012. Journal of Transplantation).
We extracted cytoplasmic proteins from frozen pancreas that have undergo different time of cold ischemia : 0, 2, 4, 6, 8, 10, 12 and 18h. Proteins were extracted using an homemade lysis buffer (20mM Tris, 1mM EDTA, 1mM EGTA, 150 mM NaCl, 0.5% Triton-X-100 and a protease inhibitor cocktail (Complete™ ULTRA Tablets, EDTA-free, glass vials Protease Inhibitor Cocktail, Roche) and were frozen at -80°C. A BCA test was performed to determine proteins concentration in our sample and 70µg of pancreatic proteins were dilute in 50µl of 1X cell extraction buffer PTR for the assay. OD were read with Glomax from Promega.
OD 450nm P-JNK1/2 JNK1/2 total P-JNK1/2 - blk JNK1/2 total - blk P-JNK/JNK
0h ischemia n1 0,219 0,477 0,151 0,426 0,355
4h ischemia n1 0,330 4,445 0,262 4,393 0,060
6h ischemia n1 0,361 4,388 0,294 4,337 0,068
8h ischemia n1 1,311 4,519 1,244 4,468 0,278
10h ischemia n1 0,476 0,555 0,409 0,504 0,811
12h ischemia n1 0,554 4,446 0,486 4,395 0,111
18h ischemia n1 0,431 0,305 0,363 0,254 1,431
0h ischemia n2 0,398 0,552 0,330 0,501 0,659
4h ischemia n2 0,382 4,434 0,315 4,383 0,072
6h ischemia n2 0,432 4,241 0,365 4,190 0,087
8h ischemia n2 0,339 0,529 0,272 0,478 0,568
10h ischemia n2 1,891 4,644 1,823 4,592 0,397
12h ischemia n2 1,034 0,342 0,966 0,291 3,319
18h ischemia n2 0,337 0,199 0,269 0,148 1,823
0h ischemia n3 0,370 4,117 0,302 4,065 0,074
4h ischemia n3 0,289 4,449 0,222 4,397 0,050
6h ischemia n3 0,309 4,545 0,241 4,494 0,054
8h ischemia n3 0,641 4,468 0,573 4,416 0,130
10h ischemia n3 0,412 1,657 0,344 1,606 0,214
12h ischemia n3 0,262 4,376 0,195 4,325 0,045
18h ischemia n3 0,257 4,425 0,189 4,374 0,043
Blank 0,068 0,051
mean P-JNK/JNK SEM
0H ischemia 0,363 0,169
4H ischemia 0,061 0,006
6H ischemia 0,069 0,010
8H ischemia 0,325 0,129
10H ischemia 0,474 0,176
12H ischemia 1,158 1,080
18H ischemia 1,099 0,540
We obtained concording results with literature : an increase of JNK activation after 8h of cold ischemia and beyond. The kit is working on rat pancreatic tissue.
Username

Mrs. Fotini MOUTH

Verified customer

Submitted Nov 24 2017

Abreviews
Abtrial CODE: ABTRIAL-EV7WD
Methods
1. Wash the 6 well plate with PBS after ultrasound exposure.
2. Lyse the cells with 0.5mL of freshly prepared Lysis buffer per well, with shaking (~300 rpm) at room temp for 10 minutes.
3. Aliquot supernatant (this is the soluble cell extract) to clean, chilled tubes on ice and store samples at -80°C. Minimize freeze/thaw cycles.
4. Determine desired number of microplate strips. Remove unused strips from frame and return to storage pouch and seal.
5. Add 50 μL/well of lysate to the microplate.
6. Add 50 μL/well Lysis Mix (negative control) and Control Lysates (positive control) to separate wells for assay controls if desired.
7. Add 50 μL/well of Antibody Mix to the wells. Cover the microplate with adhesive seal and incubate for 1 hr at room temp on a microplate shaker (400 rpm).
8. Wash wells with 350 μL/well 1X Wash Buffer (repeat 3 times). After final wash, remove any remaining wash solution from wells.
9. Add μL/well TMB and incubate in the dark on a plate shaker set to 400rpm.
10. Add 100 µL/well Stop Solution, and mix briefly (1 min) on a microplate shaker.
11. Record the OD at 450 nm.

Results
Phosphorylation of JNK was not significantly affected by Ruthenium Red after ultrasound exposure in rat BMSCs.
Username

Ms. Qianhua Gao

Verified customer

Submitted Jul 15 2015

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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