Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-PDK1 antibody [EPR19571] (ab202468)

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Overview

  • Product name

    Anti-PDK1 antibody [EPR19571]
    See all PDK1 primary antibodies

  • Description

    Rabbit monoclonal [EPR19571] to PDK1

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, ICC/IF, Flow Cyt, IPmore details

  • Species reactivity

    Reacts with: Mouse, Rat

  • Immunogen

    within Mouse PDK1 aa 400 to the C-terminus. The exact sequence is proprietary.
    Database link: Q8BFP9

  • Positive control

    • WB: L-929, NIH/3T3, F9, bEnd.3, C6 and PC-12 whole cell lysates; Mouse brain, kidney, spleen and heart lysates; Rat brain, kidney and heart lysates. ICC/IF: NIH/3T3 and RAW 264.7 cells. Flow Cyt: NIH/3T3 cells. IP: NIH/3T3 whole cell lysate.

  • General notes

     

     

     This product was previously labelled as Mitochondrial Pyruvate dehydrogenase kinase 1

     

    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 ab202468 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 1/2000. Detects a band of approximately 47 kDa (predicted molecular weight: 49 kDa).
ICC/IF 1/1000.
Flow Cyt 1/600.
IP 1/40.

Target

  • Function

    Inhibits the mitochondrial pyruvate dehydrogenase complex by phosphorylation of the E1 alpha subunit, thus contributing to the regulation of glucose metabolism.

  • Tissue specificity

    Expressed predominantly in the heart.

  • Sequence similarities

    Belongs to the PDK/BCKDK protein kinase family.
    Contains 1 histidine kinase domain.

  • Cellular localization

    Mitochondrion matrix.
  • Information by UniProt

  • Database links

    • Alternative names

      • [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial antibody
      • HGNC:8809 antibody
      • Mitochondrial pyruvate dehydrogenase kinase isoenzyme 1 antibody
      • PDH kinase 1 antibody
      • Pdk1 antibody
      • PDK1_HUMAN antibody
      • Pyruvate dehydrogenase kinase isoform 1 antibody
      • Pyruvate dehydrogenase kinase, isoenzyme 1 antibody
      see all

    Images

    • Western blot - Anti-PDK1 antibody [EPR19571] (ab202468)
      Western blot - Anti-PDK1 antibody [EPR19571] (ab202468)

      Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
      Lane 2: PDK1 knockout HAP1 whole cell lysate (20 µg)
      Lane 3: HeLa whole cell lysate (20 µg)
      Lane 4: Jurkat whole cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab202468 observed at 45 kDa. Red - loading control, ab9484, observed at 37 kDa.

      ab202468 was shown to specifically react with PDK1 in wild-type cells along with additional cross reactive bands. Signal was lost in PDK1 knockout cells. Wild-type and PDK1 knockout samples were subjected to SDS-PAGE. ab202468 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 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-PDK1 antibody [EPR19571] (ab202468)
      Immunocytochemistry/ Immunofluorescence - Anti-PDK1 antibody [EPR19571] (ab202468)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PDK1 with ab202468 at 1/1000 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 counterstain is DAPI (blue).

      Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

      The negative controls are as follows:-

      -ve control 1: ab202468 at 1/1000 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-PDK1 antibody [EPR19571] (ab202468)
      Flow Cytometry - Anti-PDK1 antibody [EPR19571] (ab202468)

      Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PDK1 with ab202468 at 1/600 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] - 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.

      Cells were permeabilised by 90% methanol-1XPBS, -20ºC, 30min.

    • Immunoprecipitation - Anti-PDK1 antibody [EPR19571] (ab202468)
      Immunoprecipitation - Anti-PDK1 antibody [EPR19571] (ab202468)

      PDK1 was immunoprecipitated from 1mg of NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate with ab202468 at 1/40 dilution.

      Western blot was performed from the immunoprecipitate using ab202468 at 1/1000 dilution.

      VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

      Lane 1: NIH/3T3 whole cell lysate, 10µg (Input).

      Lane 2: ab202468 IP in NIH/3T3 whole cell lysate.

      Lane 3:  Rabbit IgG,monoclonal -[EPR25A] - Isotype Control (ab172730) instead of ab202468 in NIH/3T3 whole cell lysate.

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

      Exposure time: 1 second.

    • Western blot - Anti-PDK1 antibody [EPR19571] (ab202468)
      Western blot - Anti-PDK1 antibody [EPR19571] (ab202468)
      All lanes : Anti-PDK1 antibody [EPR19571] (ab202468) at 1/2000 dilution

      Lane 1 : L-929 (Mouse connective tissue fibroblast cell line) whole cell lysate
      Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate
      Lane 3 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate
      Lane 4 : bEnd.3 (Mouse brain endothelioma cell line) whole cell lysate
      Lane 5 : C6 (Rat glial tumor cell line) whole cell lysate
      Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate

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

      Predicted band size: 49 kDa
      Observed band size: 47 kDa
      why is the actual band size different from the predicted?



      Blocking/Dilution buffer: 5% NFDM/TBST.

      Exposure time: Lane 1: 1 minute; Lane 3-4: 10 seconds; Lane 5/6: 8 seconds.

    • Immunocytochemistry/ Immunofluorescence - Anti-PDK1 antibody [EPR19571] (ab202468)
      Immunocytochemistry/ Immunofluorescence - Anti-PDK1 antibody [EPR19571] (ab202468)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling PDK1 with ab202468 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

      Confocal image showing cytoplasmic staining on RAW 264.7 cell line.

      The nuclear counterstain is DAPI (blue).

      Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

      The negative controls are as follows:-

      -ve control 1: ab202468 at 1/1000 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-PDK1 antibody [EPR19571] (ab202468)
      Western blot - Anti-PDK1 antibody [EPR19571] (ab202468)
      All lanes : Anti-PDK1 antibody [EPR19571] (ab202468) at 1/2000 dilution

      Lane 1 : Mouse brain lysate
      Lane 2 : Mouse kidney lysate
      Lane 3 : Mouse spleen lysate
      Lane 4 : Mouse heart lysate
      Lane 5 : Rat brain lysate
      Lane 6 : Rat kidney lysate
      Lane 7 : Rat heart 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: 49 kDa
      Observed band size: 47 kDa why is the actual band size different from the predicted?



      Blocking/Dilution buffer: 5% NFDM/TBST.

      Exposure time: Lane 1/2/3/5/6: 8 seconds; Lane 4: 3 seconds; Lane 7: 1 second.

    References

    ab202468 has not yet been referenced specifically in any publications.

    Publishing research using ab202468? Please let us know so that we can cite the reference in this datasheet.

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