Anti-DNA PKcs (phospho S2056) antibody [EPR5670]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue labelling DNA PKcs (phospho S2056) with unpurified ab124918 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

Immunocytochemistry/Immunofluorescence analysis of Jurkat cells (untreated and treated with Alkaline Phosphatase) labelling DNA PKcs (phospho S2056) with purified ab124918 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/500) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling DNA PKcs (phospho S2056) with purified ab124918 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• WB

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Western blot - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

All lanes:

Western blot - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (ab124918) at 1/1000 dilution

Lane 1:

Jurkat cell lysate, untreated at 10 µg

Lane 2:

Jurkat cell lysate, treated with alkaline phosphatase at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 469 kDa

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• WB

Lab

Western blot - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (ab124918) at 1/10000 dilution

Lane 1:

Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate - untreated at 10 µg

Lane 2:

Jurkat whole cell lysate - treated with alkaline phosphatase at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 469 kDa

Observed band size: 469 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 U2OS cells treated with Etoposide (10μM 20h) and 5 µg of ab124918 [EPR5670]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ELISA

Lab

ELISA - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

Serially diluted ab124918 was bound to immobilised phospho- or control peptides (1 microgram per mL). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.

• OI-RD Scanning

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OI-RD Scanning - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

• WB

CiteAb

Western blot - Anti-DNA PKcs (phospho S2056) antibody [EPR5670] (AB124918)

DNA PKcs (phospho S2056) western blot using anti-DNA PKcs (phospho S2056) antibody [EPR5670] ab124918. Publication image and figure legend from Xiao, X., Liang, J., et al., 2018, Nat Commun, PubMed 30337542.

ab124918 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab124918 please see the product overview.

The combination of DNA-PK inhibitors and M1 virus-induced enhanced DNA damage response. a Cells were treated with vehicle, NU7441 (1 μM), M1 (MOI = 1) or NU7441/M1 for 36 h, and comet assay was used to assess double-strand breaks (DSBs). b Quantification of tail moment from a. Box-and-whisker plots show the median (horizontal line), interquartile range (box) and min to max of the data. 62, 61, 60 and 64 cells of vehicle, NU7441, M1 and NU7441/M1 groups, were analyzed, ***p < 0.001 by Kruskal–Wallis test. c, d Immunoblots of proteins after infection with M1 virus (MOI = 1) for 0, 24, 36 and 48 h. e, f Cells were exposed to NU7441 (1 μM), M1 (MOI = 1) or a combination as indicated. p-H2AX (a marker of DNA damage response), cleaved-caspase-3 (a marker of apoptosis) and p-DNA-PKcs (target of NU7441) were determined by western blot. g, h Cells were transfected with siRNA targeting DNA-PKcs (48 h), and this was followed by M1 virus infection (MOI = 1) for 48 h. Proteins were examined by western blot

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