Overview

  • Product name
    ERK 1/2 (pT202/Y204 + Total) ELISA Kit
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    (pT202/Y204) 6 3.3%
    (Total) 6 3%
    Inter-assay
    Sample n Mean SD CV%
    (pT202/Y204) 3 1.3%
    (Total) 3 4.9%
  • 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 ERK1/2 (pT202/Y204) and ERK1/2 (Total) in vitro SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the semi-quantitative measurement of ERK1/2 (pT202/Y204) and Total ERK1/2 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-ERK (Thr202/Tyr204): 0.1 ng/mL (tested with recombinant protein), Total ERK1/2: 0.6 ng/mL (tested with recombinant protein)
    Range: Phospho-ERK (Thr202/Tyr204): 0.2-20ng/mL, Total ERK1/2: 0.6-60 ng/mL

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

Properties

Applications

Our Abpromise guarantee covers the use of ab176660 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 ERK1/2 (pT202/Y204) and ERK1/2 (Total) cell lysate dilution series. Background-subtracted data values (mean +/- SD) are graphed.

  • Example of a typical ERK1/2 (pT202/Y204) and ERK1/2 (Total) 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 ERK1/2 (pT202/Y204) are normalized and plotted.

  • Cell line analysis for Total ERK1/2 from 100 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 ERK1/2 (pT202/Y204) phosphorylation in MCF-7 cells in response to EGF treatment. MCF-7 cells were cultured in 96-well tissue culture plates, serum starved and treated (10 min) with a dose-range of EGF before cell lysis. Data from quadruplicate measurements of ERK1/2 (pT202/Y204) are plotted and compared against Total ERK1/2 protein levels. Comparative ERK1/2 (pT202/Y204) and ERK1/2 (Total) data also shown by Western Blot.

Protocols

References

This product has been referenced in:
  • Lei Q  et al. Mitochonic acid 5 activates the MAPK-ERK-yap signaling pathways to protect mouse microglial BV-2 cells against TNFa-induced apoptosis via increased Bnip3-related mitophagy. Cell Mol Biol Lett 23:14 (2018). Read more (PubMed: 29636771) »
  • Yu W  et al. Mst1 promotes cardiac ischemia-reperfusion injury by inhibiting the ERK-CREB pathway and repressing FUNDC1-mediated mitophagy. J Physiol Sci N/A:N/A (2018). Read more (PubMed: 29961191) »
See all 5 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

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 ERK is activated in rat lung at early time of cold ischemia before a decrease at longer time (Makoto I et al., 2004. Journal of Immunology).
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.
Samples p-erk erk total p-erk - blc erk total - blc p-erk/erk
0h ischemia n1 1,479432 3,998141 1,342205885 3,906141196 0,343614277
2h ischemia n1 3,240994 4,406835 3,103767484 4,314834687 0,719324774
4h ischemia n1 0,470404 4,504498 0,333177605 4,412497552 0,075507714
6h ischemia n1 0,591361 4,442557 0,45413478 4,350557189 0,104385429
8h ischemia n1 0,670239 4,686406 0,533013067 4,594405755 0,116013495
10h ischemia n1 0,355332 4,599256 0,218105779 4,507255598 0,048389929
12h ischemia n1 0,274116 4,627921 0,136890131 4,53592076 0,030179127
18h ischemia n1 0,302368 4,618768 0,1651419 4,526767589 0,036481197
0h ischemia n2 2,576674 4,429621 2,439447272 4,3376211 0,562392891
2h ischemia n2 0,926506 4,555853 0,78927955 4,463852763 0,176815767
4h ischemia n2 0,447998 4,485504 0,310771773 4,393504238 0,070734374
6h ischemia n2 0,536652 4,555587 0,399425228 4,463587256 0,089485252
8h ischemia n2 0,303704 4,665909 0,166477548 4,573908919 0,036397215
10h ischemia n2 0,446963 4,541355 0,309736955 4,449355353 0,069613895
12h ischemia n2 0,367469 4,750878 0,230242633 4,658878093 0,049420188
18h ischemia n2 0,244856 4,413728 0,107630121 4,321727509 0,024904421
0h ischemia n3 2,968467 4,173453 2,831240842 4,081452871 0,693684561
2h ischemia n3 0,32831 2,91623 0,191083917 2,824230245 0,06765876
4h ischemia n3 0,791702 4,41409 0,65447558 4,322089924 0,15142572
6h ischemia n3 0,774372 4,471827 0,637145338 4,379826758 0,145472726
8h ischemia n3 0,167049 4,455714 0,029822794 4,363714237 0,006834268
10h ischemia n3 0,208758 4,540457 0,071531248 4,448457126 0,016080013
12h ischemia n3 0,216896 4,667624 0,079669625 4,575623496 0,017411753
18h ischemia n3 0,469556 4,550441 0,332329964 4,458441381 0,074539494
Blank 0,137226 0,092
mean p-erk/erk SEM
0H ischemia 0,533 0,177
4H ischemia 0,321 0,349
6H ischemia 0,099 0,045
8H ischemia 0,113 0,029
10H ischemia 0,053 0,056
12H ischemia 0,045 0,027
18H ischemia 0,032 0,016
We obtained concording results with literature : a decrease of ERK
activation with time of cold ischemia in pancreas. Moreover, western blotting of ERK with the same samples confirm this results.

Mrs. Fotini MOUTH

Verified customer

Submitted Nov 13 2017

Abreviews
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 100 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 ERK was inhibited by Ruthenium Red after ultrasound exposure in rat BMSCs.

Ms. Qianhua Gao

Verified customer

Submitted Jul 10 2015

Very reliable, fast and easy ELISA kit.

Excellent Excellent 5/5 (Ease of Use)
Abreviews
I highly recommend this ERK SimpleStep ELISA kit. There’s a lot of ERK ELISA kit out there, but with this one you can record both total and phospho-ERK levels in less than two hours! Also it’s very reliable. I’ve used others before but they could take at least 5-6 hours to complete and I had to buy two kits one for total and one for phospho-ERK, it’s an advantage if you have few samples. I thought that I would need a lot of sample to get a decent signal in so few time, but I’ve recorded a strong signal (at least 1 OD) even with low concentrated samples (0,2mg/ml total protein), even with phospho-ERK. Stimulating with serum alone is enough to get a strong signal. On the other hand, in the blank well (both for total and phospho-ERK) the signal was almost the same as an empty well. I recommend using less sample with total ERK than with phospho-ERK or you could saturate the signal. In standard conditions, you can’t use all the plate with only one antibody, there are phospho-antibody for 60 wells and total antibody for 60 wells.

Mr. Pedro Campos

Verified customer

Submitted Dec 17 2014

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

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