Anti-DNA PKcs antibody [EPR392]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-DNA PKcs antibody [EPR392] (AB133516)

Immunocytochemistry/ Immunofluorescence analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling DNA PKcs with purified ab133516 at 1 : 200 dilution (9.3 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1 : 200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

Unknown

Western blot - Anti-DNA PKcs antibody [EPR392] (AB133516)

All lanes:

Western blot - Anti-DNA PKcs antibody [EPR392] (ab133516) at 1/1000 dilution

Lane 1:

K562 cell lysate at 10 µg

Lane 2:

Molt4 cell lysate at 10 µg

Lane 3:

MCF7 cell lysate at 10 µg

Lane 4:

SH-SY5Y cell lysate at 10 µg

Lane 5:

293T cell lysate at 10 µg

Lane 6:

PC3 cell lysate at 10 µg

Secondary

All lanes:

Goat-anti-rabbit HRP at 1/2000 dilution

Predicted band size: 469 kDa

Observed band size: 460 kDa

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• WB

Supplier Data

Western blot - Anti-DNA PKcs antibody [EPR392] (AB133516)

Blocking and diluting buffer and concentration : QD20250827IMG33.

Performed under reducing conditions.

In Western blot, ab323874 was shown to bind specifically to DNA PKcs. Target of interest was observed at 450 kDa in Calyculin A treated wild-type HAP1 cell lysates (lane 2) with no signal observed at this size in Calyculin A treated DNA PKcs knockout cell line (lane 4).

The identity of the bands lower than 450 kDa are unknown.

Lanes 1-4 on the left untreated membrane with alkaline phosphatase; Lanes 1-4 on the right treated membrane with alkaline phosphatase.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-DNA PKcs antibody [EPR392] - (ab133516) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-DNA PKcs (phospho T2609) antibody [EPR27165-32] (<a href='/en-us/products/primary-antibodies/dna-pkcs-phospho-t2609-antibody-epr27165-32-ab323874'>ab323874</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

Wild-type HAP1 treated with 100nM Calyculin A for 30min whole cell lysate at 20 µg

Lane 3:

Untreated DNA PKcs knockout HAP1 whole cell lysate at 20 µg

Lane 4:

DNA PKcs knockout HAP1 treated with 100nM Calyculin A for 30min whole cell lysate at 20 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 1/20000 dilution

Observed band size: 450 kDa,124 kDa

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Exposure time: 125s

• WB

Lab

Western blot - Anti-DNA PKcs antibody [EPR392] (AB133516)

Lane 1 : Wild type HAP1 whole cell lysate (40 μg)
Lane 2 : DNA PKcs knockout HAP1 whole cell lysate (40 μg)
Lane 3 : K562 whole cell lysate (20 μg)
Lane 4 : SHSY5Y whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab133516 observed at 460 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab133516 was shown to specifically react with DNA PKcs when DNA PKcs knockout samples were used. Wild-type and DNA PKcs knockout samples were subjected to SDS-PAGE. ab133516 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-DNA PKcs antibody [EPR392] (ab133516)

Predicted band size: 469 kDa

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• WB

Lab

Western blot - Anti-DNA PKcs antibody [EPR392] (AB133516)

Anti-PRKDC antibody [EPR392] (ab133516) staining at 1/2000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab133516 was shown to bind specifically to PRKDC. 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 [EPR392] (ab133516) at 1/2000 dilution

Lanes 1 - 4:

Western blot 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>)

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

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• WB

Lab

Western blot - Anti-DNA PKcs antibody [EPR392] (AB133516)

All lanes:

Western blot - Anti-DNA PKcs antibody [EPR392] (ab133516) at 1/5000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 15 µg

Lane 2:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysates at 15 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 1/20000 dilution

Predicted band size: 469 kDa

Observed band size: 460 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-DNA PKcs antibody [EPR392] (AB133516)

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.