Validated using a knockout cell lineRecombinantRabMAb

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

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR18702] to PI 3 Kinase p85 alpha
  • Suitable for: WB, ICC/IF, Flow Cyt, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

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 antibody
    • PI3 kinase p85 subunit alpha antibody
    • PI3-kinase regulatory subunit alpha antibody
    • PI3-kinase subunit p85-alpha antibody
    • PI3K antibody
    • PI3K p85 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)
    All lanes : Anti-PI 3 Kinase p85 alpha antibody [EPR18702] (ab191606) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : PIK3R1 knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 84 kDa
    Observed band size: 90 kDa
    why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab191606 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab191606 was shown to react with PI 3 Kinase p85 alpha in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265116 (knockout cell lysate ab257029) was used. Wild-type HeLa and PIK3R1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab191606 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 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)

    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.

     

     

  • 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.

  • 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). 

  • 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.

  • 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).

  • 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.

  • 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).

  • 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).

  • 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).

  • 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 Detection Reagent (HRP) (ab131366), was used for detection 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.

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

References (81)

Publishing research using ab191606? Please let us know so that we can cite the reference in this datasheet.

ab191606 has been referenced in 81 publications.

  • He S  et al. Ferulic Acid Ameliorates Lipopolysaccharide-Induced Barrier Dysfunction via MicroRNA-200c-3p-Mediated Activation of PI3K/AKT Pathway in Caco-2 Cells. Front Pharmacol 11:376 (2020). PubMed: 32308620
  • Li Z  et al. Compression stress induces nucleus pulposus cell autophagy by inhibition of the PI3K/AKT/mTOR pathway and activation of the JNK pathway. Connect Tissue Res N/A:1-13 (2020). PubMed: 32180463
  • Xia W & Jie W ZEB1-AS1/miR-133a-3p/LPAR3/EGFR axis promotes the progression of thyroid cancer by regulating PI3K/AKT/mTOR pathway. Cancer Cell Int 20:94 (2020). PubMed: 32231464
  • Pan X  et al. Exosomal MicroRNA-221-3p Confers Adriamycin Resistance in Breast Cancer Cells by Targeting PIK3R1. Front Oncol 10:441 (2020). PubMed: 32426266
  • Wu S  et al. High expression of UNC5B enhances tumor proliferation, increases metastasis, and worsens prognosis in breast cancer. Aging (Albany NY) 12:17079-17098 (2020). PubMed: 32902412
View all Publications for this product

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Western blot abreview for Anti-PI 3 Kinase p85 alpha antibody [EPR18702]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Mouse Tissue lysate - whole (Aging mouse kidney)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
Aging mouse kidney
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
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Submitted Jul 10 2020

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