Anti-DNA PKcs antibody [Y393]

• ICC/IF

Lab

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.

• IHC-P

Lab

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.

• IHC-P

Lab

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.

• IHC-P

Lab

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.

• ICC/IF

Lab

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.

• WB

Lab

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] (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 (<a href='/en-us/products/cell-lines/human-prkdc-knockout-a549-cell-line-ab276100'>ab276100</a>)

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

Predicted band size: 469 kDa

Observed band size: 450 kDa

false

• WB

Lab

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

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• WB

Lab

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.

All lanes:

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

Predicted band size: 469 kDa

false

• WB

Lab

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

false

• ChIC/CUT&RUN-seq

Supplier Data

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 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.

• WB

Unknown

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

false