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  1. Link

    dna-pkcs-antibody-y393-ab32566.pdf

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Epigenetics and Nuclear Signaling DNA / RNA DNA Damage & Repair Non Homol. End Joining
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-DNA PKcs antibody [Y393] (ab32566)

  • Datasheet
Reviews (13)Q&A (1)References (29)

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Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
  • Anti-DNA PKcs antibody [Y393] (ab32566)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y393] to DNA PKcs
  • Suitable for: WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human

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Overview

  • Product name

    Anti-DNA PKcs antibody [Y393]
    See all DNA PKcs primary antibodies
  • Description

    Rabbit monoclonal [Y393] to DNA PKcs
  • Host species

    Rabbit
  • Specificity

    This antibody is specific for DNA PKcs. It may also detect the splice isoform 2. 

    Mouse and rat species are recommended based on WB results, we do not guarantee IHC-P for mouse and rat.

  • Tested applications

    Suitable for: WB, IHC-P, ICC/IFmore details
    Unsuitable for: Flow Cyt or IP
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat, Armenian hamster
  • Immunogen

    Synthetic peptide within Human DNA PKcs aa 4050 to the C-terminus (C terminal). The exact sequence is proprietary.
    Database link: P78527

  • Positive control

    • WB: K562 cell lysate. ICC/IF: Hela cells. IHC-P: Human breast carcinoma tissue slides.
  • General notes

    Mouse and rat samples are recommended based on WB results.

    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.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

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

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    Y393
  • Isotype

    IgG
  • Research areas

    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Damage & Repair
    • Non Homol. End Joining
    • Epigenetics and Nuclear Signaling
    • DNA / RNA
    • DNA Damage & Repair
    • DNA Damage Response
    • DNA Damage Recognition
    • Neuroscience
    • Processes

Associated products

  • Alternative Versions

    • Anti-DNA PKcs antibody [Y393] - BSA and Azide free (ab174575)
    • HRP Anti-DNA PKcs antibody [Y393] (ab195537)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Recombinant Protein

    • Recombinant Human DNA PKcs protein (Tagged) (ab204152)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32566 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 (10)
1/1000 - 1/10000. Detects a band of approximately 460 kDa (predicted molecular weight: 469 kDa).

For unpurified, use 1/1000 - 1/2000.

IHC-P
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified, use 1/5.  

 

ICC/IF (2)
1/100.

For unpurified, use 1/10.

Notes
WB
1/1000 - 1/10000. Detects a band of approximately 460 kDa (predicted molecular weight: 469 kDa).

For unpurified, use 1/1000 - 1/2000.

IHC-P
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified, use 1/5.  

 

ICC/IF
1/100.

For unpurified, use 1/10.

Application notes
Is unsuitable for Flow Cyt or IP.

Target

  • Function

    Serine/threonine-protein kinase that acts as a molecular sensor for DNA damage. Involved in DNA nonhomologous end joining (NHEJ) required for double-strand break (DSB) repair and V(D)J recombination. Must be bound to DNA to express its catalytic properties. Promotes processing of hairpin DNA structures in V(D)J recombination by activation of the hairpin endonuclease artemis (DCLRE1C). The assembly of the DNA-PK complex at DNA ends is also required for the NHEJ ligation step. Required to protect and align broken ends of DNA. May also act as a scaffold protein to aid the localization of DNA repair proteins to the site of damage. Found at the ends of chromosomes, suggesting a further role in the maintenance of telomeric stability and the prevention of chromosomal end fusion. Also involved in modulation of transcription. Recognizes the substrate consensus sequence [ST]-Q. Phosphorylates 'Ser-139' of histone variant H2AX/H2AFX, thereby regulating DNA damage response mechanism. Phosphorylates DCLRE1C, c-Abl/ABL1, histone H1, HSPCA, c-jun/JUN, p53/TP53, PARP1, POU2F1, DHX9, SRF, XRCC1, XRCC1, XRCC4, XRCC5, XRCC6, WRN, MYC and RFA2. Can phosphorylate C1D not only in the presence of linear DNA but also in the presence of supercoiled DNA. Ability to phosphorylate p53/TP53 in the presence of supercoiled DNA is dependent on C1D.
  • Sequence similarities

    Belongs to the PI3/PI4-kinase family.
    Contains 1 FAT domain.
    Contains 1 FATC domain.
    Contains 2 HEAT repeats.
    Contains 1 PI3K/PI4K domain.
    Contains 3 TPR repeats.
  • Post-translational
    modifications

    Phosphorylated upon DNA damage, probably by ATM or ATR. Autophosphorylated on Thr-2609, Thr-2638 and Thr-2647. Thr-2609 is a DNA damage-inducible phosphorylation site (inducible with ionizing radiation, IR). Autophosphorylation induces a conformational change that leads to remodeling of the DNA-PK complex, requisite for efficient end processing and DNA repair.
    S-nitrosylated by GAPDH.
  • Cellular localization

    Nucleus.
  • Target information above from: UniProt accession P78527 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 5591 Human
    • Entrez Gene: 19090 Mouse
    • Entrez Gene: 360748 Rat
    • Omim: 600899 Human
    • SwissProt: P78527 Human
    • SwissProt: P97313 Mouse
    • Unigene: 491682 Human
    • Unigene: 71 Mouse
    • Alternative names

      • DNA dependent protein kinase catalytic subunit antibody
      • DNA PK catalytic subunit antibody
      • DNA-dependent protein kinase catalytic subunit antibody
      • DNA-PK catalytic subunit antibody
      • DNA-PKcs antibody
      • DNAPK antibody
      • DNAPK catalytic subunit antibody
      • DNPK 1 antibody
      • DNPK1 antibody
      • Hyper radiosensitivity of murine scid mutation, complementing 1 antibody
      • Hyperradiosensitivity complementing 1, mouse, homolog of 1 antibody
      • HYRC 1 antibody
      • HYRC antibody
      • HYRC1 antibody
      • IMD26 antibody
      • p350 antibody
      • p460 antibody
      • PKRDC antibody
      • PRKDC antibody
      • PRKDC_HUMAN antibody
      • Protein Kinase DNA Activated Catalytic Polypeptide antibody
      • XRCC 7 antibody
      • XRCC7 antibody
      see all

    Images

    • Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
      Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: DNA PKcs knockout HAP1 cell lysate (20 µg)
      Lane 3: K562 cell lysate (20 µg)
      Lane 4: SH-SY5Y cell lysate (20 µg)
      Lanes 1 - 4: Merged signal (red and green). Green - ab32566 observed at 470 kDa. Red - loading control, ab7291, observed at 52 kDa.


      ab32566 was shown to specifically react with DNA PKCs in wild-type HAP1 cells. No band was observed when DNA PKcs knockout samples were examined. Wild-type and DNA PKCs knockout samples were subjected to SDS-PAGE. ab32566 and ab7291 (loading control to alpha tubulin) were diluted 1/1000 and 1/10,000 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 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)

      Immunohistochemical staining of paraffin embedded human tonsil with purified ab32566 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

    • Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)
      Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)

      Immunofluorescent staining of HeLa cells (fixed with 4% PFA) with purified ab32566 at a dilution of 1/100. An Alexa Fluor® 555 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200. The panel on the right shows the DAPI counter-staining.

    • Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
      Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
      All lanes : Anti-DNA PKcs antibody [Y393] (ab32566) at 1/7300 dilution (purified)

      Lane 1 : MOLT4 cell lysate
      Lane 2 : HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 469 kDa
      Observed band size: 460 kDa why is the actual band size different from the predicted?



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)

      Immunohistochemical staining of paraffin embedded human tonsil with unpurified ab32566 at a working dilution of 1 in 5. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

    • Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)
      Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [Y393] (ab32566)

      Immunofluorescent staining of HeLa cells (fixed with 4% PFA) with unpurified ab32566 at a dilution of 1/10. An Alexa Fluor® 555 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200. The panel on the right shows the DAPI counter-staining.

    • Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
      Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
      All lanes : Anti-DNA PKcs antibody [Y393] (ab32566) at 1/1000 dilution (unpurified)

      Lane 1 : MOLT4 cell lysate
      Lane 2 : HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 469 kDa
      Observed band size: 460 kDa why is the actual band size different from the predicted?



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs antibody [Y393] (ab32566)

      Unpurified ab32566, at a 1/50 dilution, staining DNA PKcs in paraffin embedded human breast carcinoma tissue by immunohistochemistry.

    • Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
      Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
      Anti-DNA PKcs antibody [Y393] (ab32566) at 1/1000 dilution (unpurified) + K562 cell lysate.

      Predicted band size: 469 kDa
      Observed band size: 460 kDa why is the actual band size different from the predicted?

    • Anti-DNA PKcs antibody [Y393] (ab32566)
      Anti-DNA PKcs antibody [Y393] (ab32566)

    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

    Datasheets and documents

    • Datasheet download

      Download

    References (29)

    Publishing research using ab32566? Please let us know so that we can cite the reference in this datasheet.

    ab32566 has been referenced in 29 publications.

    • Zhang W  et al. Lycorine hydrochloride suppresses stress-induced premature cellular senescence by stabilizing the genome of human cells. Aging Cell 20:e13307 (2021). PubMed: 33455051
    • Guo Z  et al. LncRNA linc00312 suppresses radiotherapy resistance by targeting DNA-PKcs and impairing DNA damage repair in nasopharyngeal carcinoma. Cell Death Dis 12:69 (2021). PubMed: 33431817
    • Fan J  et al. Oncolytic herpes simplex virus and temozolomide synergistically inhibit breast cancer cell tumorigenesis in vitro and in vivo. Oncol Lett 21:99 (2021). PubMed: 33376532
    • Bosch-Guiteras N  et al. Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs. Genome Res 31:461-471 (2021). PubMed: 33574136
    • Liu Y  et al. Gut Microbiota Dysbiosis Accelerates Prostate Cancer Progression Through Increased LPCAT1 Expression and Enhanced DNA Repair Pathways. Front Oncol 11:679712 (2021). PubMed: 34221998
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-10 of 14 Abreviews or Q&A

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa)
    Loading amount
    20 µg
    Specification
    HeLa
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Read More

    Abcam user community

    Verified customer

    Submitted Dec 30 2010

    Immunocytochemistry/ Immunofluorescence abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (breast cancer cell)
    Specification
    breast cancer cell
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5% triton X100
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 09 2008

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - nuclear (Vascular smooth muscle cells)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    25 µg
    Specification
    Vascular smooth muscle cells
    Blocking step
    Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 29 2017

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HEK293)
    Loading amount
    200000 cells
    Specification
    HEK293
    Gel Running Conditions
    Reduced Denaturing (5% gel)
    Blocking step
    Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Read More

    Abcam user community

    Verified customer

    Submitted Feb 02 2010

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (A549 tumour cell line)
    Loading amount
    50 µg
    Specification
    A549 tumour cell line
    Treatment
    100IU bleomycin for 2h
    Gel Running Conditions
    Reduced Denaturing (3-8% tris acetate gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 27 2009

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (mouse muscle)
    Loading amount
    100 µg
    Specification
    mouse muscle
    Gel Running Conditions
    Reduced Denaturing (6% gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 14 2009

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Armenian Hamster Tissue lysate - whole (Liver tissue)
    Loading amount
    50 µg
    Specification
    Liver tissue
    Gel Running Conditions
    Reduced Denaturing (6% gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 14 2009

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    African Green Monkey Cell lysate - whole cell (COS-7 cell line)
    Loading amount
    20 µg
    Specification
    COS-7 cell line
    Gel Running Conditions
    Reduced Denaturing (6% gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Sep 11 2009

    Western blot abreview for Anti-DNA PKcs antibody [Y393]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Rat Cell lysate - whole cell (C6 Cell line)
    Loading amount
    25 µg
    Specification
    C6 Cell line
    Gel Running Conditions
    Reduced Denaturing (6% gel)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Jun 11 2008

    Question

    Purchasing 1549-1 lot YG021806C and 1579-1 lot YC120107R
    The Epitomics datasheets says the lot numbers can be used to find the isotype control of the correct concentration.
    Please confirm which isotype controls.

    Read More

    Abcam community

    Verified customer

    Asked on Oct 25 2012

    Answer

    Thank you for contacting us and your interest in our products.

    Epitomics did offer isotype controls specific for different lots of antibody purchased. This was providing the antibody at the same concentration as the antibody to be tested. We do not currently have this available from our catalogue. We do however have a rabbit monoclonal isotype control. This can be viewed from the following link:

    www.abcam.com/ab125938

    This is provided at a concentration of 0.2 mg/mL.

    I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

    Read More

    Abcam Scientific Support

    Answered on Oct 25 2012

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