Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody (ab138482)

Rabbit polyclonal Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody. Validated in WB, ELISA, IHC and tested in Mouse, Human. Cited in 1 publication(s). Independently reviewed in 1 review(s).

Overview

  • Product name

    Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody
    See all Erk1 (pT202/pY204) + Erk2 (pT185/pY187) primary antibodies
  • Description

    Rabbit polyclonal to Erk1 (pT202/pY204) + Erk2 (pT185/pY187)
  • Host species

    Rabbit
  • Specificity

    Please note - This antibody detects all 4 modifications (T185 + Y187 in ERK2 and T202 + Y204 in ERK1) by Elisa, however we only detect 2 modifications (T185 in ERK2 and T202 in ERK1) by western blot.
  • Tested applications

    Suitable for: ELISA, WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide corresponding to Human Erk1 (pT202/pY204) + Erk2 (pT185/pY187) aa 150-250 conjugated to keyhole limpet haemocyanin.
    Database link: P27361

  • Positive control

    • This antibody gave a positive signal in PDGF stimulated (200ul) NIH3T3 whole cell lysates. This antibody gave a positive result in IHC in the following FFPE tissue: Human breast Adenocarcinoma

Properties

Applications

Our Abpromise guarantee covers the use of ab138482 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
ELISA Use a concentration of 1 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 41, 43 kDa (predicted molecular weight: 41,43 kDa).
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Images

  • IHC image of Erk1 (pT202/pY204) + Erk2 (pT185/pY187) staining in Human breast Adenocarcinoma formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab138482, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • All lanes : Anti-Erk1 (pT202/pY204) + Erk2 (pT185/pY187) antibody (ab138482) at 1 µg/ml

    Lane 1 : PDGF-Stimulated NIH 3T3 Whole Cell Lysate
    Lane 2 : PDGF-Stimulated NIH 3T3 Whole Cell Lysate with Immunising peptide (Phosphorylation T 202 + Phosphorylation Y 204) at 1 µg/ml
    Lane 3 : PDGF-Stimulated NIH 3T3 Whole Cell Lysate with Non-modified Control peptide at 1 µg/ml
    Lane 4 : PDGF-Stimulated NIH 3T3 Whole Cell Lysate with Single Mod Control peptide (Phosphorylation T 202) at 1 µg/ml
    Lane 5 : PDGF-Stimulated NIH 3T3 Whole Cell Lysate with Single Mod Control peptide (Phosphorylation Y 204) at 1 µg/ml

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 41,43 kDa
    Observed band size: 41 + 43 kDa
    why is the actual band size different from the predicted?


    Exposure time: 2 minutes


    Please note - This antibody detects all 4 modifications (T185 + Y187 in ERK2 and T202 + Y204 in ERK1) by Elisa, however we only detect 2 modifications (T185 in ERK2 and T202 in ERK1) by western blot.

    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab138482 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406

  • Western blot analysis of ERK1 + ERK2 (phospho T185 + Y187 + T202 + Y204) using ab138482 at 1/1000.

    Lane 1 - Bacterial expressed ERK2 negative control.

    Lane 2 - Bacterial expressed ERK2 with MEK1 R4F, positive control.

    Lane 3 - PP2A treated phosphorylated ERK2.

    Lane 4 - PTP1 U323 treated phosphorylated ERK2. M, Molecular weight marker.

    ERK2 is phosphorylated at T183 and Y185. This antibody detects the dual phospho, and the pT183 forms. After treating the pT183 pY185 ERK2 with the serine/threonine phosphatase PP2A the antibody does not detect it. After treating the pT183 pY185 ERK2 with the tyrosine phosphatase PTPi U323 the antibody does detect it.

    A IRDye® 680-conjugated donkey anti-rabbit IgG (H+L) polyclonal (1/10000) was used as the secondary antibody.

    See Abreview

  • ab138482 was tested using an Indirect ELISA approach. The wells were coated with peptide (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary Ab was then added at a dilution range of 1- 0.00025µg/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature.

References

This product has been referenced in:

  • Chalenko Y  et al. Hepatoprotective Activity of InlB321/15, the HGFR Ligand of Bacterial Origin, in CCI4-Induced Acute Liver Injury Mice. Biomedicines 7:N/A (2019). Read more (PubMed: 30979058) »
  • Liu Y  et al. KRASG12 mutant induces the release of the WSTF/NRG3 complex, and contributes to an oncogenic paracrine signaling pathway. Oncotarget 7:53153-53164 (2016). Read more (PubMed: 27449290) »
See all 2 Publications for this product

Customer reviews and Q&As

Application
Western blot
Loading amount
0.1 µg
Gel Running Conditions
Non-reduced Denaturing (10% SDS-PAGE)
Sample
Rat Recombinant protein (Expressed in Origami(DE3) E. coli)
Specification
Expressed in Origami(DE3) E. coli
Treatment
PP2A, PTP1 U323 2 hrs at 30C
Blocking step
Odyssey Blocking Buffer in TBS-T as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 4°C

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Verified customer

Submitted Sep 30 2014

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