Name: Anti-DNA PKcs antibody [Y393]
SKU: ab32566
Rating: 5 (13 reviews)

Knockout Tested Rabbit Recombinant Monoclonal DNA PKcs antibody. Suitable for ChIC/CUT&RUN-seq, WB, ICC/IF, IHC-P and reacts with Human samples. Cited in 47 publications.

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Images

Western blot - Anti-DNA PKcs antibody [Y393] (AB32566), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ChIC/CUT&RUN-seq	IP	Flow Cyt	WB	ICC/IF	IHC-P
Human	Tested	Not recommended	Not recommended	Tested	Tested	Tested
Mouse	Predicted	Not recommended	Not recommended	Predicted	Predicted	Predicted
Rat	Predicted	Not recommended	Not recommended	Predicted	Predicted	Predicted
Armenian hamster	Predicted	Not recommended	Not recommended	Predicted	Predicted	Predicted

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 5 µg	Notes -

Predicted
Predicted

Species	Dilution info	Notes
Species Mouse, Rat, Armenian hamster	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human	Dilution info -	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Mouse	Dilution info -	Notes -
Species Rat	Dilution info -	Notes -
Species Armenian hamster	Dilution info -	Notes -

Not recommended
Not recommended

Species	Dilution info	Notes
Species Human, Mouse, Rat, Armenian hamster	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/1000 - 1/10000	Notes For unpurified, use 1/1000 - 1/2000.

Predicted
Predicted

Species	Dilution info	Notes
Species Mouse, Rat, Armenian hamster	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/100	Notes For unpurified, use 1/10.

Predicted
Predicted

Species	Dilution info	Notes
Species Mouse, Rat, Armenian hamster	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/50	Notes For unpurified, use 1/5. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Predicted
Predicted

Species	Dilution info	Notes
Species Mouse, Rat, Armenian hamster	Dilution info -	Notes -

Associated Products

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Target data

See full target information PRKDC

Function

Serine/threonine-protein kinase that acts as a molecular sensor for DNA damage (PubMed:11955432, PubMed:12649176, PubMed:14734805, PubMed:33854234). Involved in DNA non-homologous end joining (NHEJ) required for double-strand break (DSB) repair and V(D)J recombination (PubMed:11955432, PubMed:12649176, PubMed:14734805, PubMed:33854234, PubMed:34352203). Must be bound to DNA to express its catalytic properties (PubMed:11955432). Promotes processing of hairpin DNA structures in V(D)J recombination by activation of the hairpin endonuclease artemis (DCLRE1C) (PubMed:11955432). Recruited by XRCC5 and XRCC6 to DNA ends and is required to (1) protect and align broken ends of DNA, thereby preventing their degradation, (2) and sequester the DSB for repair by NHEJ (PubMed:11955432, PubMed:12649176, PubMed:14734805, PubMed:15574326, PubMed:33854234). Act as a scaffold protein to aid the localization of DNA repair proteins to the site of damage (PubMed:11955432, PubMed:12649176, PubMed:14734805, PubMed:15574326). The assembly of the DNA-PK complex at DNA ends is also required for the NHEJ ligation step (PubMed:11955432, PubMed:12649176, PubMed:14734805, PubMed:15574326). Found at the ends of chromosomes, suggesting a further role in the maintenance of telomeric stability and the prevention of chromosomal end fusion (By similarity). Also involved in modulation of transcription (PubMed:11955432, PubMed:12649176, PubMed:14734805, PubMed:15574326). As part of the DNA-PK complex, involved in the early steps of ribosome assembly by promoting the processing of precursor rRNA into mature 18S rRNA in the small-subunit processome (PubMed:32103174). Binding to U3 small nucleolar RNA, recruits PRKDC and XRCC5/Ku86 to the small-subunit processome (PubMed:32103174). Recognizes the substrate consensus sequence [ST]-Q (PubMed:11955432, PubMed:12649176, PubMed:14734805, PubMed:15574326). Phosphorylates 'Ser-139' of histone variant H2AX, thereby regulating DNA damage response mechanism (PubMed:14627815, PubMed:16046194). Phosphorylates ASF1A, DCLRE1C, c-Abl/ABL1, histone H1, HSPCA, c-jun/JUN, p53/TP53, PARP1, POU2F1, DHX9, FH, SRF, NHEJ1/XLF, XRCC1, XRCC4, XRCC5, XRCC6, WRN, MYC and RFA2 (PubMed:10026262, PubMed:10467406, PubMed:11889123, PubMed:12509254, PubMed:14599745, PubMed:14612514, PubMed:14704337, PubMed:15177042, PubMed:1597196, PubMed:16397295, PubMed:18644470, PubMed:2247066, PubMed:2507541, PubMed:26237645, PubMed:26666690, PubMed:28712728, PubMed:29478807, PubMed:30247612, PubMed:8407951, PubMed:8464713, PubMed:9139719, PubMed:9362500). Can phosphorylate C1D not only in the presence of linear DNA but also in the presence of supercoiled DNA (PubMed:9679063). Ability to phosphorylate p53/TP53 in the presence of supercoiled DNA is dependent on C1D (PubMed:9363941). Contributes to the determination of the circadian period length by antagonizing phosphorylation of CRY1 'Ser-588' and increasing CRY1 protein stability, most likely through an indirect mechanism (By similarity). Plays a role in the regulation of DNA virus-mediated innate immune response by assembling into the HDP-RNP complex, a complex that serves as a platform for IRF3 phosphorylation and subsequent innate immune response activation through the cGAS-STING pathway (PubMed:28712728). Also regulates the cGAS-STING pathway by catalyzing phosphorylation of CGAS, thereby impairing CGAS oligomerization and activation (PubMed:33273464). Also regulates the cGAS-STING pathway by mediating phosphorylation of PARP1 (PubMed:35460603).

Alternative names

HYRC, HYRC1, PRKDC, DNA-dependent protein kinase catalytic subunit, DNA-PK catalytic subunit, DNA-PKcs, DNPK1, p460

Recommended products

Knockout Tested Rabbit Recombinant Monoclonal DNA PKcs antibody. Suitable for ChIC/CUT&RUN-seq, WB, ICC/IF, IHC-P and reacts with Human samples. Cited in 47 publications.

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Clone number

Y393

Purification technique

Affinity purification Protein A

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.

Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Mouse and rat samples are recommended based on WB results.

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 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 batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

DNA-dependent protein kinase catalytic subunit often abbreviated as DNA-PKcs or PKcs is an important component in the cellular machinery responsible for DNA repair. This protein weighs approximately 470 kDa and is predominantly expressed in various tissue types with higher levels found in lymphoid tissues. The DNA-PKcs functions as a serine/threonine protein kinase and becomes active upon binding to DNA contributing to its role in maintaining genome integrity.

Biological function summary

DNA-PKcs plays an essential role in the non-homologous end joining (NHEJ) repair pathway which repairs double-strand breaks in DNA. DNA-PKcs forms a complex with the Ku70/80 heterodimer serving as a critical component of the DNA repair mechanism. This complex recognizes DNA ends facilitates their synapsis and activates other enzymes involved in ligating the broken DNA strands therefore promoting cellular survival following DNA damage.

Pathways

DNA-PKcs deeply integrates into the DNA damage response pathway. It collaborates closely with proteins such as ATM (ataxia-telangiectasia mutated) to sense DNA damage and initiate repair. DNA-PKcs is involved in V(D)J recombination vital for the generation of diverse antibodies in immune cells. Its activity is essential for coordinating with other proteins including XRCC4 and Ligase IV to ensure efficient DNA repair processes are executed.

Associated diseases and disorders

Defects in DNA-PKcs are linked to severe combined immunodeficiency (SCID) due to its central role in V(D)J recombination. Dysfunction in its pathway also relates to cancer where impaired DNA repair mechanisms substantially increase genomic instability leading to tumorigenesis. DNA-PKcs interacts with other proteins like BRCA1 and p53 in tumors where its overexpression or mutations can contribute to resistance against radiation and chemotherapeutic agents. These interactions highlight the importance of DNA-PK inhibitors as potential therapeutic strategies in oncology.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

11 product images

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, Anti-alpha Tubulin antibody [DM1A] - 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 Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (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.
All lanes: Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
Predicted band size: 469 kDa
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.
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)
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
All lanes: Western blot - Anti-DNA PKcs antibody [Y393] (ab32566) at 1/7300 dilution
Lane 1: MOLT4 cell lysate at 20 µg
Lane 2: HeLa cell lysate at 20 µg
Secondary
All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 469 kDa
Observed band size: 460 kDa
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.
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.
Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST
All lanes: Western blot - Anti-DNA PKcs antibody [Y393] (ab32566) at 1/1000 dilution
Lane 1: MOLT4 cell lysate at 20 µg
Lane 2: HeLa cell lysate at 20 µg
Secondary
All lanes: HRP goat anti-rabbit (H+L) at 1/1000 dilution
Predicted band size: 469 kDa
Observed band size: 460 kDa
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)
All lanes: Western blot - Anti-DNA PKcs antibody [Y393] (ab32566) at 1/1000 dilution
All lanes: K562 cell lysate.
Predicted band size: 469 kDa
Observed band size: 460 kDa
Western blot - Anti-DNA PKcs antibody [Y393] (ab32566)
False colour image of Western blot: Anti-DNA PKcs antibody [Y393] staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32566 was shown to bind specifically to DNA PKcs. A band was observed at 450 kDa in wild-type A549 cell lysates with no signal observed at this size in PRKDC knockout cell line. To generate this image, wild-type and PRKDC knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-DNA PKcs antibody [Y393] (ab32566) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: PRKDC knockout A549 cell lysate at 20 µg
Lane 2: Western blot - Human PRKDC knockout A549 cell line (Human PRKDC knockout A549 cell line ab276100)
Lane 3: K562 cell lysate at 20 µg
Lane 4: HDLM-2 cell lysate at 20 µg
Secondary
All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 469 kDa
Observed band size: 450 kDa
ChIC/CUT&RUN sequencing - Anti-DNA PKcs antibody [Y393] (ab32566)
ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2 x 10^5 U2OS cells and 5 µg of ab ab32566 [Y393]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.

Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.