Overview

  • Product name

    AKT/MAPK Signaling Pathway Antibody Cocktail
  • Sample type

    Cell culture extracts, Tissue Extracts, Cell Lysate, Human PBMC Lysate, Mouse muscle extract, Tissue Homogenate, Nuclear Extracts
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Product overview

    ab151279 is a Western Blot Antibody Cocktail that contains 5 Rabbit antibodies targeting the phosphorylation status of 4 signaling proteins, AKT1 phospho S473, RPS6 phospho S235/236, ERK1/2 phospho Y204/187 and RKS1p90 phospho S380. Along with the total levels of 1 loading control protein, Rab11.

  • Notes

    The cocktail targets downstream effectors of two important pro-survival pathways: the PI3K/PTEN/Akt/mTOR pathway and the Raf/MEK/ERK pathway, both of which are regulated by Ras. PI3K downstream effectors targeted in this cocktail are AKT1 phospho S473 and RPS6 phospho S235/236, whereas the downstream effectors of the MEK pathway are ERK1/2 phospho Y204/197 and p90RSK phospho S380. These two pathways are known for promoting cell growth, regulating apoptosis, chemotherapeutic drug resistance and cellular senescence. Both of these pathways affect protein translation by complex interactions regulating mTORC1/2 complexes; they regulate each other as well as other pathways such as Wnt/B-catenin, Jak/STAT, NF-κB and TGF-B.

  • Tested applications

    Suitable for: WBmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab151279 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
WB Use at an assay dependent concentration.

Suggested working concentration: 1/250

Images

  • Performed under reducing conditions; Samples loaded at 30 µg/lane. Membrane blocking and secondary antibody incubation performed in 5% milk/PBST. Primary antibody incubation performed in 5% BSA/PBST.

    Lane 1: Marker
    Lane 2: A431 lysate starved
    Lane 3: A431 lysate starved treated with 100 ng/mL of EGF for 30 min
    Lane 4: A431 lysate starved and pretreated for 2 hours with 10µM U0126 (ab120241)
    Lane 5: A431 lysate starved and pretreated for 2 hours with 30µM Ly294002 (ab120243)

    Primary: AKT/MAPK signaling pathway antibody cocktail at 1/250 dilution

    Secondary: HRP conjugated Goat anti-Rabbit secondary antibody at 1:10,000 dilution.

    Observed bands:
    RSK1p90 (pS380) band size: 83 kDa
    AKT1 (pS473) band size: 56 kDa
    ERK1 (pY204) and ERK2 (pY187) band size: 43 kDa
    RPS6 (pS235/236) band size: 31 kDa
    Rab11a band size: 24 kDa
    Unknown band: 130kDa
  • Developed using ECL; performed under reducing conditions; exposure time: 5 mins; samples run on a 4-20% gradient gel.
    Membrane blocking (2hrs RT) and secondary antibody incubation (2hrs RT) steps must be done in 5% milk, 1X PBS, 0.1% TWEEN-20.
    Primary antibody incubation (24 hrs 4°C) must be done in 5% BSA, 1X PBS, 0.1% TWEEN-20. Secondary: HRP conjugated Goat anti-Rabbit secondary antibody at 1:10,000 dilution.
    Samples:
    Lane 1: Marker
    Lanes 2-6: A431 cell lysate at 30 µg
    Primary:
    Lane 1: none
    Lane 2: Anti-RSK1p90 (pS380)
    Lane 3: Anti-AKT1 (pS473)
    Lane 4: Anti-ERK1 (pY204) and ERK2 (pY187)
    Lane 5: Anti-RPS6 (pS235/236)
    Lane 6: Anti-Rab11a
    Observed bands:
    RSK1p90 (pS380) band size: 83 kDa
    AKT1 (pS473) band size: 56 kDa
    ERK1 (pY204) and ERK2 (pY187) band size: 43 kDa
    RPS6 (pS235/236) band size: 31 kDa
    Rab11a band size: 24 kDa
  • All lysates loaded at 30 µg/lane.
    Membrane blocking (2hrs RT) and secondary antibody incubation (2hrs RT) steps must be done in 5% milk, 1X PBS, 0.1% TWEEN-20.
    Primary antibody incubation (24 hrs 4°C) must be done in 5% BSA, 1X PBS, 0.1% TWEEN-20, at 1/250 dilution.
    Secondary: IR690 conjugated Goat anti-Rabbit secondary antibody at 1/10,000 dilution
    Lane 1: Marker
    Lane 2: A431 cells starved O/N
    Lane 3: A431 cells starved O/N treated with 100 ng/mL of EGF for 30 min
    Lane 4: A431 cells starved O/N and pretreated for 2 hours with 10 µM UO126 prior to EGF treatment
    Lane 5: A431 cells starved O/N and pretreated for 2 hours with 30 µM Ly294002 prior to EGF treatment
  • Developed using the ECL technique; performed under reducing conditions; exposure time: 5 mins; samples run on a 4-20% gradient gel.
    Membrane blocking (2hrs RT) and secondary antibody incubation (2hrs RT) steps must be done in 5% milk, 1X PBS, 0.1% TWEEN-20.
    Primary antibody incubation (24 hrs 4°C) must be done in 5% BSA, 1X PBS, 0.1% TWEEN-20.
    Lane 1: Marker.
    Lane 2: NIH3T3 cells starved overnight and treated with 50ng/mL of PDGF for 30 minutes lysate – 30 µg.
    Primary: AKT/MAPK signaling pathway antibody cocktail at 1/250 dilution.
    Secondary: HRP conjugated Goat anti-Rabbit secondary antibody at 1/10,000 dilution.
    Observed bands:
    RSK1p90 (pS380) band size: 83 kDa
    AKT1 (pS473) band size: 56 kDa
    ERK1 (pY204) and ERK2 (pY187) band size: 43 kDa
    RPS6 (pS235/236) band size: 31 kDa
    Rab11a band size: 24 kDa
    Unknown band: 130kDa

References

This product has been referenced in:

  • Zhou QL  et al. FPR1 gene silencing suppresses cardiomyocyte apoptosis and ventricular remodeling in rats with ischemia/reperfusion injury through the inhibition of MAPK signaling pathway. Exp Cell Res 370:506-518 (2018). Read more (PubMed: 30031130) »
  • Zhang HN  et al. Berberine promotes nerve regeneration through IGFR-mediated JNK-AKT signal pathway. Mol Med Rep 18:5030-5036 (2018). Read more (PubMed: 30272344) »
See all 2 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Human cardiomyocytes blot

Good Good 4/5 (Ease of Use)
Abreviews
Western blot using 5-15% gradient gel
sample volume is 30µG/lane
Blocking: 5%milk in TBS+0.1% Tween
Primary Antibody: 1:1000 5%milk in TBS+0.1% Tween
Incubation at 4 degree overnight.
2ndry antibody: Goat anti Rabbit, HRP-conjugated
Incubation for 1 hour at room temperature.
Developed using ECL for 3 mins.
Observed bands:
Rab11a: 24Kda
RPS6: 31Kda
ERK1/2: 43Kda
pS473: 56-58Kda
pS380: 75Kda

Abcam user community

Verified customer

Submitted Jul 04 2019

Mouse Cardiomyocytes Blot

Excellent Excellent 5/5 (Ease of Use)
Abreviews
Electrophoresis performed under reducing conditions on a gradient gel 4-20%.
Samples loaded at 20 µg/lane. Membrane blocking and secondary antibody incubation performed in 5% milk/PBST. Primary antibody incubation performed in 5% BSA/PBST.
Lane 1: Marker
Lane 2 Empty
Lane 3: WT Control cardiomyocytes
Lane 4: WT cardiomyocytes stimulated with ISO for 30 minutes
Primary antibody: AKT/MAPK signaling pathway antibody cocktail at 1/500 dilution
Secondary antibody: HRP conjugated Goat anti-Rabbit secondary antibody at 1:10,000 dilution.
Developed using ECL for 5 minutes.
Observed bands:
RSK1p90 (pS380) band size: 83 kDa
AKT1 (pS473) band size: 56 kDa
ERK1 (pY204) and ERK2 (pY187) band size: 43 kDa
RPS6 (pS235/236) band size: 31 kDa
Rab11a band size: 24 kDa

Abcam user community

Verified customer

Submitted May 30 2019

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