Validated using a knockout cell line
Recombinant
RabMAb

Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

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Overview

  • Product name
    Anti-PI 3 Kinase p85 alpha antibody [EPR18702]
    See all PI 3 Kinase p85 alpha primary antibodies
  • Description
    Rabbit monoclonal [EPR18702] to PI 3 Kinase p85 alpha
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Rat PI 3 Kinase p85 alpha aa 600-700. The exact sequence is proprietary.
    Database link: Q63787

  • Positive control
    • WB: Human PI3K p85 alpha full length recombinant protein; Human fetal liver, fetal heart and fetal kidney lysates; HepG2, MCF7, Raji, Jurkat, C6, RAW 264.7, PC-12 and NIH/3T3 whole cell lysates; Mouse brain, heart, kidney and spleen lysates; Rat brain, heart, kidney and spleen lysates. ICC/IF: HepG2 and NIH/3T3 cells. Flow Cyt: NIH/3T3 cells; IP: MCF7 whole cell lysate.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab191606 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 1/1000. Detects a band of approximately 85,46 kDa (predicted molecular weight: 84 kDa).
ICC/IF 1/250.
Flow Cyt 1/150.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP 1/50.

Target

  • Function
    Binds to activated (phosphorylated) protein-Tyr kinases, through its SH2 domain, and acts as an adapter, mediating the association of the p110 catalytic unit to the plasma membrane. Necessary for the insulin-stimulated increase in glucose uptake and glycogen synthesis in insulin-sensitive tissues.
  • Tissue specificity
    Isoform 2 is expressed in skeletal muscle and brain, and at lower levels in kidney and cardiac muscle. Isoform 2 and isoform 4 are present in skeletal muscle (at protein level).
  • Sequence similarities
    Belongs to the PI3K p85 subunit family.
    Contains 1 Rho-GAP domain.
    Contains 2 SH2 domains.
    Contains 1 SH3 domain.
  • Domain
    The SH3 domain mediates the binding to CBLB, and to HIV-1 Nef.
  • Post-translational
    modifications
    Polyubiquitinated in T-cells by CBLB; which does not promote proteasomal degradation but impairs association with CD28 and CD3Z upon T-cell activation.
    Phosphorylated. Dephosphorylated by PTPRJ.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • GRB1 antibody
    • p85 alpha antibody
    • p85 antibody
    • P85A_HUMAN antibody
    • Phosphatidylinositol 3 kinase associated p 85 alpha antibody
    • Phosphatidylinositol 3 kinase regulatory 1 antibody
    • Phosphatidylinositol 3 kinase, regulatory subunit, polypeptide 1 (p85 alpha) antibody
    • Phosphatidylinositol 3-kinase 85 kDa regulatory subunit alpha antibody
    • Phosphatidylinositol 3-kinase regulatory subunit alpha antibody
    • Phosphoinositide 3 kinase, regulatory subunit 1 (alpha) antibody
    • PI3 kinase p85 subunit alpha antibody
    • PI3-kinase regulatory subunit alpha antibody
    • PI3-kinase subunit p85-alpha antibody
    • PI3K antibody
    • PI3K regulatory subunit alpha antibody
    • Pik3r1 antibody
    • PtdIns 3 kinase p85 alpha antibody
    • PtdIns-3-kinase regulatory subunit alpha antibody
    • PtdIns-3-kinase regulatory subunit p85-alpha antibody
    see all

Images

  • Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: PIK3R1 knockout HAP1 whole cell lysate (20 µg)
    Lane 3: Jurkat whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab191606 observed at 90 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab191606 was shown to specifically react with PIK3R1 when PIK3R1 knockout samples were used. Wild-type and PIK3R1 knockout samples were subjected to SDS-PAGE. Ab191606 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

     

     

  • Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    All lanes : Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/20000 dilution

    Lane 1 : Human PI3K p85 alpha full length recombinant protein
    Lane 2 : Human PI3K p85 beta full length recombinant protein

    Lysates/proteins at 0.01 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 84 kDa


    Exposure time: 5 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    Human PI3K p85 alpha full length recombinant protein contain aa1-724 with a His-Tag® (Cat#ab84769). Human PI3K p85 beta full length recombinant protein contain aa1-728 with a His-Tag® (Cat#ab125568). 

  • Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    All lanes : Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

    Lane 1 : Human fetal liver lysate
    Lane 2 : Human fetal heart lysate
    Lane 3 : Human fetal kidney lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

    Predicted band size: 84 kDa
    Observed band size: 46,85 kDa (why is the actual band size different from the predicted?)


    Exposure time: 3 minutes


    Blocking/Dilution buffer: 5% NFDM/TBST.

    The molecular weight observed is consistent with what have been described in the literatures (PMID: 8921377, 12649157).

  • Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    All lanes : Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

    Lane 1 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
    Lane 2 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
    Lane 3 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
    Lane 4 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 84 kDa
    Observed band size: 46,85 kDa (why is the actual band size different from the predicted?)



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time:  Lane 1 and 2: 10 seconds;  Lane 3 and 4: 3 seconds.

    The molecular weight observed is consistent with what have been described in the literatures (PMID: 8921377, 12649157).

  • Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Western blot - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    All lanes : Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

    Lane 1 : Mouse brain lysate
    Lane 2 : Mouse heart lysate
    Lane 3 : Mouse kidney lysate
    Lane 4 : Mouse spleen lysate
    Lane 5 : Rat brain lysate
    Lane 6 : Rat heart lysate
    Lane 7 : Rat kidney lysate
    Lane 8 : Rat spleen lysate
    Lane 9 : C6 (Rat glial tumor cell line) whole cell lysate
    Lane 10 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
    Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
    Lane 12 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 84 kDa
    Observed band size: 46,85 kDa (why is the actual band size different from the predicted?)



    Blocking/Dilution buffer: 5% NFDM/TBST.

    Exposure time: Lane 1-8: 3 minutes; Lane 9-12: 10 seconds.

    The molecular weight observed is consistent with what have been described in the literatures (PMID: 8921377, 12649157).

  • Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling PI3K p85 with ab191606 at 1/250 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HepG2 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291)  at 1/1000 dilution and Goat Anti-Mouse IgG (AlexaFluor®594) preadsorbed (ab150120)  at 1/1000 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab191606 at 1/500 dilution followed by ab150120  at 1/1000 dilution.
    -ve control 2: ab7291  at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

  • Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Immunocytochemistry/ Immunofluorescence - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 100% Methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PI3K p85 with ab191606 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody - Loading Control (ab7291) at  1/1000 dilution and Goat Anti-Mouse IgG  (AlexaFluor®594) preadsorbed (ab150120) at 1/1000 dilution (red).
    The negative controls are as follows:
    -ve control 1: ab191606 at 1/500 dilution followed by ab150120  at 1/1000 dilution.
    -ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

  • Flow Cytometry - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Flow Cytometry - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

    Overlay histogram showing HepG2 cells fixed in 4% PFA and stained with ab191606 at a dilution of 1/80 (red line). The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit at a dilution of 1/500. Rabbit monoclonal IgG (ab172730) was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

  • Flow Cytometry - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Flow Cytometry - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

    Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PI3K p85 with ab191606 at 1/150 dilution (red) compared with a Rabbit IgG,monoclonal -  Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor® 488) at 1/500 dilution was used as the secondary antibody.

  • Immunoprecipitation - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)
    Immunoprecipitation - Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606)

    PI3K p85 was immunoprecipitated from 1mg of MCF7 (Human breast adenocarcinoma cell line) whole cell lysate with ab191606 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab191606 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/10000 dilution.

    Lane 1: MCF7 whole cell lysate, 10µg (Input).

    Lane 2: ab191606 IP in MCF7 whole cell lysate.

    Lane 3: Rabbit IgG, monoclonal - Isotype Control (ab172730) instead of ab191606 in MCF7 whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

    Exposure time: 5 seconds.

References

This product has been referenced in:
  • Zhuo J  et al. Evaluation of type 2 diabetic mellitus animal models via interactions between insulin and mitogen-activated protein kinase signaling pathways induced by a high fat and sugar diet and streptozotocin. Mol Med Rep 17:5132-5142 (2018). Read more (PubMed: 29393432) »
  • Liu S & Liu B Overexpression of Nitrogen Permease Regulator Like-2 (NPRL2) Enhances Sensitivity to Irinotecan (CPT-11) in Colon Cancer Cells by Activating the DNA Damage Checkpoint Pathway. Med Sci Monit 24:1424-1433 (2018). Read more (PubMed: 29519997) »

See all 10 Publications for this product

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