Anti-DNA PKcs (phospho S2056) antibody - ChIP Grade (ab18192)

Overview

  • Product nameAnti-DNA PKcs (phospho S2056) antibody - ChIP Grade
    See all DNA PKcs primary antibodies
  • Description
    Rabbit polyclonal to DNA PKcs (phospho S2056) - ChIP Grade
  • SpecificityThis antibody specifically recognizes a band at ~460 kDa in HeLa cells that have been treated with ionizing radiation, that is not detected in untreated cells. This band can also be competed away by the immunizing modified peptide, but not the unmodified peptide containing the same amino acid sequence. All batches of this antibody are screened in ELISA and show high titres against the immunising peptide. Reactivity with the unmodified DNA PKcs peptide is minimal. We now predict that this antibody will cross react with mouse DNA PKcs, as the mouse sequence has been more extensively reviewed on uniprot (P97313), now indicating that mouse DNA PKcs S2053, which corresponds to human S2056, is also phosphorylated. We welcome any feedback from researchers who have used this antibody with mouse samples.
  • Tested applicationsSuitable for: Flow Cyt, IHC-P, WB, ChIP, ELISA, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 2050 - 2150 of Human DNA PKcs.

    (Peptide available as ab20406.)

  • Positive control
    • This antibody gave a positive signal in Hela cells which have been treated with 20-40 mU/ml bleomycin for 48hrs. ICC/IF: Hela cells UV treated

Properties

Applications

Our Abpromise guarantee covers the use of ab18192 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 1/100. PubMed: 18559621ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 460 kDa (predicted molecular weight: 460 kDa).Can be blocked with Human DNA PKcs (phospho S2056) peptide (ab20406).
ChIP Use at an assay dependent concentration. PubMed: 20714222
ELISA Use at an assay dependent concentration.

Direct ELISA with serially diluted ab18192 (0.2-1000 ng x mL-1), bound to immobilised phospho or control peptides (1 µg x mL-1).

ICC/IF Use a concentration of 1 - 5 µg/ml.

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.
  • 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
    • 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

Anti-DNA PKcs (phospho S2056) antibody - ChIP Grade images

  • ICC/IF image of ab18192 stained UV treated HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18192 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • All lanes : Anti-DNA PKcs (phospho S2056) antibody - ChIP Grade (ab18192) at 1 µg/ml

    Lane 1 : HeLa Gamma Irradiated Whole Cell Lysate Pack (ab13823) at 20 µg
    Lane 2 : 20ug of untreated HeLa cell extract
    Lane 3 : HeLa Gamma Irradiated Whole Cell Lysate Pack (ab13823) at 20 µg with Human DNA PKcs (phospho S2056) peptide (ab20406) at 1 µg/ml
    Lane 4 : 20ug of untreated HeLa cell extract with Human DNA PKcs (phospho S2056) peptide (ab20406) at 1 µg/ml
    Lane 5 : HeLa Gamma Irradiated Whole Cell Lysate Pack (ab13823) at 20 µg with Human DNA PKcs peptide (ab20407) at 1 µg/ml
    Lane 6 : 20ug of untreated HeLa cell extract with Human DNA PKcs peptide (ab20407) at 1 µg/ml

    Secondary
    Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/10000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 460 kDa
    Observed band size : 460 kDa
    Additional bands at : 270 kDa (possible cleavage fragment,cross reactivity).

    ab18192 specifically recognizes a band at ~460 kDa corresponding to DNA PKcs in HeLa cells that have been treated with ionizing radiation (lane 1). This band is not detected in untreated cells (lane 2). The activity of the antibody is quenched by the addition of the immunizing (modified) peptide, ab20406 (lanes 3) but not the unmodified peptide, ab20407 (lane 5).

    For the ab13823 irradiated HeLa cell lysate, the 4 hour post-treatment extract was used.

  • ab18192 staining DNA PKcs in Human aorta tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 100% blocking agent for 20 minutes; antigen retrieval was enzymatic using Proteinase K. Samples were incubated with primary antibody (1/350 in diluent) for 15 hours at 4°C. A biotinylated Goat anti-rabbit polyclonal (1/800) was used as the secondary antibody.

    See Abreview

  • Serially diluted ab18192 was bound to immobilised DNA PKcs phospho peptide (2052 - 2062; P-S2056) or DNA PKcs control peptide (2052 - 2062; both at  1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.

  • Overlay histogram showing Jurkat cells stained with ab18192 (blue line). The cells were prepared as follows: Spin down, wash in FACS buffer 1x, fix, wash 2x, and stain with primary antibody overnight. Buffer was was 0.1% sodium azide with FBS in PBS. Cells fixed with paraformaldehyde and then permeabilized with Triton X-100 and NP40. Cell population was gated by isolating cell population from plot of SSC-A / FSA-A. The cells were then incubated the antibody at 1/100 for 24 hours. The secondary antibody used was FITC-conjugated Goat anti-Rabbit polyclonal at 1/100 dilution. Isotype control antibody (red line) was used under the same conditions.

    See Abreview

  • ab18192 staining DNA PKcs (phospho S2056) in leukaemia cell lines by Immunocytochemistry/ Immunofluorescence.

    Cells were either untreated (control) or treated with ionizing radiation (IR) or ultrasound (US). Following treatment, cells were fixed with paraformaldehyde and permabilized/blocked with 2% BSA/0.05% Triton X-100/Tris-buffered saline. Samples were incubated with primary antibody (1/600 in diluent) for 2 hours. An AlexaFluor®555-conjugated anti-rabbit IgG (1/400) was used as the secondary antibody.
  • All lanes : Anti-DNA PKcs (phospho S2056) antibody - ChIP Grade (ab18192) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Hela Whole Cell Lysate - Bleomycin Treated (20U/ml)
    Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Human DNA PKcs (phospho S2056) peptide (ab20406) at 1 µg/ml
    Lane 4 : Hela Whole Cell Lysate - Bleomycin Treated (20U/ml) with Human DNA PKcs (phospho S2056) peptide (ab20406) at 1 µg/ml
    Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Human DNA PKcs peptide (ab20407) at 1 µg/ml
    Lane 6 : Hela Whole Cell Lysate - Bleomycin Treated (20U/ml) with Human DNA PKcs peptide (ab20407) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 460 kDa
    Observed band size : 460 kDa
    Additional bands at : 117 kDa,150 kDa,270 kDa,50 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 15 minutes

References for Anti-DNA PKcs (phospho S2056) antibody - ChIP Grade (ab18192)

This product has been referenced in:
  • DeLoughery Z  et al. DNA double-strand breaks by Cr(VI) are targeted to euchromatin and cause ATR-dependent phosphorylation of histone H2AX and its ubiquitination. Toxicol Sci 143:54-63 (2015). Read more (PubMed: 25288669) »
  • Wilson JE  et al. Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt. Nat Med N/A:N/A (2015). Read more (PubMed: 26107252) »

See all 38 Publications for this product

Product Wall

Abcam has not validated the combination of species/application used in this Abreview.
Application Flow Cytometry
Sample Mouse Cell (bone marrow)
Permeabilization Yes - 0.5% Triton 100X
Gating Strategy live single cell
Specification bone marrow
Preparation Cell harvesting/tissue preparation method: flushing
Sample buffer: PBS
Fixation Methanol
Username

Abcam user community

Verified customer

Submitted Aug 11 2016

Application Western blot
Loading amount 10 µg
Gel Running Conditions Reduced Denaturing (6%)
Sample Human Cell lysate - whole cell (Hela cells)
Specification Hela cells
Treatment Irradiation 10 Gy
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Dr. Remi Buisson

Verified customer

Submitted Nov 03 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 20 minute(s) · Concentration: 5% · Temperature: RT°C
Sample Human Cell (T98G Human brain glioblastoma)
Specification T98G Human brain glioblastoma
Permeabilization Yes - 0.1% v/v Triton X-100 pH 7.4 for 5 min at RT
Fixative Paraformaldehyde
Username

Dr. Dimitra Kalamida

Verified customer

Submitted Dec 12 2013

Thank you for your enquiry regarding ab18192 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

As our Abpromise indicates, in the event that a p...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Human Tissue sections (Human Aorta)
Specification Human Aorta
Fixative Paraformaldehyde
Antigen retrieval step Enzymatic - Buffer/Enzyme Used: Proteinase-K, Dako
Permeabilization No
Blocking step Protein Block (DAKO) as blocking agent for 20 minute(s) · Concentration: 100%
Username

Abcam user community

Verified customer

Submitted May 02 2012

Thank you for your reply.
I would advice that you use 8% polyacrylamide gels and then use the following transfer buffer: 48mM Tris, 39mM Glycine, 0.1% SDS and 10% MeOH. I would also do your transfer overnight at 4C (25mV).
Please let me know ...

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Thank you for contacting Abcam.
I am sorry that you are experiencing problems with ab18192 in western blot. To be able to help you further, could you please provide me with some more information:
1 - What sample type are you using and from wh...

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Thank you for letting me know that the new lot of ab18192 has proved to be successful. Please let me know if there is anything else I can help you with.

Thank you for your reply. I have sent you a new aliquot of ab18192 from a different lot as you requested. Hopefully this new lot will be more successful for you. Your new order number is**********. Please let me know if there is anything else I can hel...

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Thank you for providing that information. I only have one further question; how much total protein are you loading into your wells? I ask becasue based on the Abreviews on our website, to get a good signal, you need to load 20-30ug of protein per lane....

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