Anti-DNA PKcs (phospho T2609) antibody (ab4194)

Overview

  • Product nameAnti-DNA PKcs (phospho T2609) antibodySee all DNA PKcs primary antibodies ...
  • Description
    Rabbit polyclonal to DNA PKcs (phospho T2609)
  • SpecificityThis phospho specific polyclonal antibody reacts with phosphorylated pT2609 of human DNA PKcs. Reactivity with non-phosphorylated human DNA PKcs is minimal by ELISA. This antibody recognises phospho T2609 DNA PKcs as demonstrated: IPed DNA PKcs (using another DNA PKcs antibody) blotted with the pT2609 antibody shows an inducible signal at the correct height after DNA damage. However, unfortunately the antibody picks up an IR-inducible signal in cells lacking DNA-PKcs (MO59J cells) at the same size as DNA-PKcs. We believe that this is 53BP1 which runs at the same size as DNA-PKcs and is also phosphorylated in an IR-dependent manner. Similar results were seen with another phospho DNA-PKcs antibody indicating that this result is general to antibodies to this phospho site, rather than specific to this antibody. This antibody may be a useful marker for DNA damage. It may also still be useful in the study of phospho DNA PKcs T2609.
  • Tested applicationsIHC-P, ICC/IF, WB more details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide: PMFVEpTQASQG, corresponding to amino acids 2604-2614 of Human DNA PKcs (phosphorylated form of human DNA PKcs at the pT2609 residue).

  • Positive control
    • IHC-P: FFPE human tonsil tissue sections.

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage bufferPreservative: 0.01% Sodium Azide
    Constituents: 0.15M Sodium Chloride, 0.02M Potassium Phosphate. pH 7.2
  • Concentration information loading...
  • PurityImmunogen affinity purified
  • Purification notesThe product was affinity purified from monospecific antiserum by immunoaffinity purification. Antiserum was first purified against the phosphorylated form of the immunizing peptide. The resultant affinity purified antibody was then cross-adsorbed against the non-phosphorylated form of the immunizing peptide. The product is sterile filtered.
  • Clonality Polyclonal
  • IsotypeIgG
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab4194 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use at an assay dependent concentration.
WB 1/1000. Detects a band of approximately >400 kDa (predicted molecular weight: 469 kDa). Works in WB after IP with another DNA PKcs antibody. When used without IP, this antibody also detects another phospho protein, believed to be 53BP1.Caution: when used on whole cell extracts (without IP) this antibody detects several background bands that are not seen with nuclear extracts.

Target

  • FunctionSerine/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 similaritiesBelongs 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 localizationNucleus.
  • 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
  • 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
    • HYRC 1 antibody
    • HYRC antibody
    • HYRC1 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

Anti-DNA PKcs (phospho T2609) antibody images

  • IHC image of DNA PKcs staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab4194, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.



  • Predicted band size : 469 kDa

    Western blot on cell lysates using ab4194 at 1/1000.

    Lane 1: Fus1 untreated
    Lane 2: Fus1 IR (20Gy, 4h)
    Lane 3: Fus1 DNAPK inhibitor + IR
    Lane 4: MO59J (DNAPK-) untreated
    Lane 5: MO59J IR
    Lane 6: Fus1 untreated
    Lane 7: Fus1 IR (20Gy, 4h)
    Lane 8: Fus1 DNAPK inhibitor + IR
    Lane 9: MO59J untreated
    Lane 10: MO59J IR

    Lane 1-5: Nuclear Lysate
    Lane 6-10: Whole Cell Lysate

    MO59J is a cell line that lacks DNA-PKcs.
    FUS1 is the matched cell line complemented with a chromosomal fragment containing the DNA-PKcs gene.

    As discussed above, we believe the upper bands represent a mixture of DNA PKcs phosphorylation and 53BP1 phosphorylation.

    Western blot on cell lysates using ab4194 at 1/1000.

    Lane 1: Fus1 untreated
    Lane 2: Fus1 IR (20Gy, 4h)
    Lane 3: Fus1 DNAPK inhibitor + IR
    Lane 4: MO59J (DNAPK-) untreated
    Lane 5: MO59J IR
    Lane 6: Fus1 untreated
    Lane 7: Fus1 IR (20Gy, 4h)
    Lane 8: Fus1 DN

References for Anti-DNA PKcs (phospho T2609) antibody (ab4194)

This product has been referenced in:
  • Chen X  et al. Suberoylanilide hydroxamic acid induces hypersensitivity to radiation therapy in acute myelogenous leukemia cells expressing constitutively active FLT3 mutants. PLoS One 8:e84515 (2013). Human . Read more (PubMed: 24367670) »
  • Bencokova Z  et al. ATM activation and signaling under hypoxic conditions. Mol Cell Biol 29:526-37 (2009). WB ; Human . Read more (PubMed: 18981219) »

See all 6 Publications for this product

Product Wall

Thank you very much for your interest in ab18356, ab4194, and ab97611.

To our knowledge, ab18356, ab4194, and ab97611 have not been tested in mouse. We don't have smaller sizes of our antibodies, but I can offer a discount off a future purch...

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Application Western blot
Sample Human Cell lysate - whole cell (primary human fibroblasts)
Loading amount 200000 cells
Specification primary human fibroblasts
Treatment 12 Gy (IR), 30 min. post IR cells were collected
Gel Running Conditions Reduced Denaturing (6)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Dr. Nicole Verkaik

Verified customer

Submitted Feb 23 2010

Thank you for your enquiry. To our knowledge, this antibody has yet to be tested in IP. All tested applications are specified on Abcam product datasheets. If you decide to go ahead and purchase this product, please let us know how you get on by submitt...

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Thank you for your phone call. Below is our protocol for Western blotting with phospho-proteins. If you have any additional questions, please let us know. WESTERN BLOTTING OF PHOSPHO-PROTEINS Homogenize the cells or tissue of interest in lysis bu...

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Thank you for your reply. When this antibody is used without IP, it detects not only DNA-PK but another phospho protein which is probably 53BP1. Therefore we would strongly suggest carrying out the Western blot after IP if you plan to detect DNA-PK...

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Thank you for your comments which we take very seriously. I discussed this with a colleague and we do think that the antibody is specific. It was tested in parallel with another phospho DNA-PKcs antibody, which gave the same result, indicating that thi...

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Thank you for your enquiry and patience. There are no publications yet that we know of that have used ab4194. The Western blot image on the datasheet is our own data. Regarding the signal that is seen in the MO59J cells, we have the following note on t...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"