Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-PKR antibody [Y117] (ab32506)

Reviews (3)Q&A (1)References (15)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y117] to PKR
  • Suitable for: Flow Cyt, WB, IHC-P, ICC/IF, IP
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-PKR antibody [Y117]
    See all PKR primary antibodies

  • Description

    Rabbit monoclonal [Y117] to PKR

  • Host species

    Rabbit

  • Specificity

    The antibody does not cross-react with other GCN2 family members.

  • Tested applications

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

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Synthetic peptide within Human PKR aa 500-600 (C terminal). The exact sequence is proprietary.

  • Positive control

    • WB: MCF-7, HEK293 and HeLa whole cell lysate (ab150035) and human liver carcinoma tissue lysate. ICC/IF: Wild type HAP1, MCF-7 and HeLa cells. IHC-P: Human liver carcinoma and colon carcinoma tissue.

  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

    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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    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

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.

  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    Y117

  • Isotype

    IgG

  • Research areas

Applications

Our Abpromise guarantee covers the use of ab32506 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
Flow Cyt Use at an assay dependent concentration.
WB 1/5000 - 1/20000. Detects a band of approximately 68 kDa (predicted molecular weight: 62 kDa).
IHC-P 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/100.

For unpurified use at 1/250 - 1/500
IP 1/80 - 1/100.

Target

  • Function

    Following activation by double-stranded RNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the translation initiation factor EIF2S1, which leads to an inhibition of the initiation of protein synthesis. Double-stranded RNA is generated during the course of a viral infection.

  • Sequence similarities

    Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. GCN2 subfamily.
    Contains 2 DRBM (double-stranded RNA-binding) domains.
    Contains 1 protein kinase domain.

  • Post-translational
    modifications

    Autophosphorylated on several Ser and Thr residues. Autophosphorylation of Thr-451 is dependent on Thr-446 and is stimulated by dsRNA binding and dimerization. Autophosphorylation apparently leads to the activation of the kinase.
  • Information by UniProt

  • Database links

    • Alternative names

      • Double stranded RNA activated protein kinase; antibody
      • E2AK2_HUMAN antibody
      • eIF-2A protein kinase 2 antibody
      • EIF2AK1 antibody
      • EIF2AK2 antibody
      • Eukaryotic translation initiation factor 2 alpha kinase 2 antibody
      • Eukaryotic translation initiation factor 2-alpha kinase 2 antibody
      • HGNC:9437 antibody
      • Interferon induced double stranded RNA activated protein kinase antibody
      • Interferon inducible elF2 alpha kinase antibody
      • Interferon inducible RNA dependent protein kinase antibody
      • Interferon-induced, double-stranded RNA-activated protein kinase antibody
      • Interferon-inducible RNA-dependent protein kinase antibody
      • MGC126524 antibody
      • P1/eIF-2A protein kinase antibody
      • P1/eIF2A protein kinase antibody
      • p68 kinase antibody
      • PKR antibody
      • PPP1R83 antibody
      • PRKR antibody
      • Protein kinase interferon inducible double stranded RNA dependent antibody
      • Protein kinase RNA activated antibody
      • Protein kinase RNA-activated antibody
      • Protein phosphatase 1 regulatory subunit 83 antibody
      • Serine/threonine protein kinase TIK antibody
      • Tyrosine protein kinase EIF2AK2 antibody
      see all

    Images

    • Western blot - Anti-PKR antibody [Y117] (ab32506)
      Western blot - Anti-PKR antibody [Y117] (ab32506)

      Lanes 1, 3 and 5: PKR knockout HAP1 cell lysate (20 µg)
      Lanes 2, 4 and 6: Wild-type HAP1 cell lysate (20 µg)
      Lanes 1 and 2: Green signal from target - ab32506 observed at 62 kDa
      Lanes 3 and 4: Red signal from loading control - ab8245 observed at 37 kDa
      Lanes 5 and 6: Merged (red and green) signal
      ab32506 was shown to specifically react with PKR when PKR knockout samples were used. Wild-type and PKR knockout samples were subjected to SDS-PAGE. ab32506 and ab8245 (loading control to GAPDH) were diluted 1/10 000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (ab32506)
      Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (ab32506)

      ab32506 staining PKR in wild-type HAP1 cells (top panel) and PKR knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32506 at 1/400 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

      This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 4% formaldehyde (10 min).


      Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

    • Western blot - Anti-PKR antibody [Y117] (ab32506)
      Western blot - Anti-PKR antibody [Y117] (ab32506)
      All lanes : Anti-PKR antibody [Y117] (ab32506) at 1/20000 dilution

      Lane 1 : MCF-7 (human breast carcinoma) whole cell lysates
      Lane 2 : HEK293 (human embryonic kidney) whole cell lysates

      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: 62 kDa
      Additional bands at: 68 kDa. We are unsure as to the identity of these extra bands.



      Purified format.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKR antibody [Y117] (ab32506)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKR antibody [Y117] (ab32506)

      ab32506 staining PKR in human liver carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

      Negative control 1: PBS in place of primary antibody.

    • Flow Cytometry - Anti-PKR antibody [Y117] (ab32506)
      Flow Cytometry - Anti-PKR antibody [Y117] (ab32506)

      Flow Cytometry analysis of MCF-7 (human breast carcinoma) cells labeling PKR with purified ab32506 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

    • Western blot - Anti-PKR antibody [Y117] (ab32506)
      Western blot - Anti-PKR antibody [Y117] (ab32506)
      Anti-PKR antibody [Y117] (ab32506) at 1/10000 dilution (unpurified) + MCF-7 cell lysate

      Predicted band size: 62 kDa
      Observed band size: 68 kDa
      why is the actual band size different from the predicted?

    • Immunoprecipitation - Anti-PKR antibody [Y117] (ab32506)
      Immunoprecipitation - Anti-PKR antibody [Y117] (ab32506)

      ab32506 immunoprecipitating PKR. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/40 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

      Lane 1: HEK293 (human embryonic kidney) whole cell lysate (10ug)
      Lane 2: HEK293 (human embryonic kidney) whole cell lysate
      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab32506 in HEK293 (human embryonic kidney) whole cell lysate

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKR antibody [Y117] (ab32506)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKR antibody [Y117] (ab32506)

      Immunohistochemical analysis of paraffin-embedded human colon carcinoma using unpurified ab32506 at 1/100 dilution.

    • Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (ab32506)
      Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (ab32506)

      ab32506 staining PKR in MCF-7 (human breast carcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody. ab7291 and ab150120 were used as counterstains for primary antibody ab32506 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

      Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
      Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

    • Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (ab32506)
      Immunocytochemistry/ Immunofluorescence - Anti-PKR antibody [Y117] (ab32506)

      Immunofluorescent staining of HeLa cells using unpurified ab32506 at 1/250 dilution.

    • Anti-PKR antibody [Y117] (ab32506)
      Anti-PKR antibody [Y117] (ab32506)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References (15)

    Publishing research using ab32506? Please let us know so that we can cite the reference in this datasheet.

    ab32506 has been referenced in 15 publications.

    • Piazzi M  et al. Expression of the double-stranded RNA-dependent kinase PKR influences osteosarcoma attachment independent growth, migration, and invasion. J Cell Physiol 235:1103-1119 (2020). PubMed: 31240713
    • Li Y  et al. eIF2a-CHOP-BCl-2/JNK and IRE1a-XBP1/JNK signaling promote apoptosis and inflammation and support the proliferation of Newcastle disease virus. Cell Death Dis 10:891 (2019). PubMed: 31767828
    • Jasenosky LD  et al. The FDA-Approved Oral Drug Nitazoxanide Amplifies Host Antiviral Responses and Inhibits Ebola Virus. iScience 19:1279-1290 (2019). PubMed: 31402258
    • Yeung ATY  et al. A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection. mBio 10:N/A (2019). PubMed: 31594818
    • Suzuki S  et al. Dental pulp cell-derived powerful inducer of TNF-a comprises PKR containing stress granule rich microvesicles. Sci Rep 9:3825 (2019). PubMed: 30846715
    View all Publications for this product

    Customer reviews and Q&As

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    1-4 of 4 Abreviews or Q&A

    Western blot abreview for Anti-PKR antibody [Y117]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HEK293)
    Loading amount
    20 µg
    Specification
    HEK293
    Gel Running Conditions
    Non-reduced Denaturing (10% gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Ms. Samantha Johnston

    Verified customer

    Submitted Sep 27 2011

    Western blot abreview for Anti-PKR antibody [Y117]

     Average
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa Cells)
    Loading amount
    20 µg
    Specification
    HeLa Cells
    Treatment
    DMSO, SiRNA Luciferase
    Gel Running Conditions
    Non-reduced Denaturing (10)
    Blocking step
    Milk as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Abcam user community

    Verified customer

    Submitted Aug 23 2011

    Western blot abreview for Anti-PKR antibody [Y117]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (primary human myoblast)
    Loading amount
    35 µg
    Specification
    primary human myoblast
    Gel Running Conditions
    Reduced Denaturing (4-12%)
    Blocking step
    odyssey blocking buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: RT°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 23 2010

    Question

    Please advise what is the concentration of this ab ( ug/ul)?

    Read More
    Answer

    Thank you for your enquiry. I can confirm that the concentration of this product is 0.157 mg/ml. Should you require any other information, please do not hesitate to contact me.

    Read More

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