Anti-Ku70 antibody [EPR4027]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ku70 antibody [EPR4027] (AB92450)

Paraffin embedded Human testis tissue (A) or Human tonsil tissue (B) were labelled with unpurified ab92450 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ku70 antibody [EPR4027] (AB92450)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ku70 with purified ab92450 at 1/20 dilution (5μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Ku70 antibody [EPR4027] (AB92450)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ku70 with purified ab92450 at 1/250 dilution (0.46 μg/ml). Cells were fixed in 100% Methanol and permeabilized with None. 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 (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Ku70 antibody [EPR4027] (AB92450)

Immunofluorescence staining of HeLa cells using unpurified ab92450 at 1/100 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Ku70 antibody [EPR4027] (AB92450)

Overlay histogram showing HeLa cells stained with unpurified ab92450 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab92450, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ku70 antibody [EPR4027] (AB92450)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon carcinoma tissue sections labeling Ku70 with purified ab92450 at 1/250 dilution (0.46 μg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 1 (pH 6.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody.

Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

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Immunoprecipitation - Anti-Ku70 antibody [EPR4027] (AB92450)

ab92450 (purified ) at 1/20 dilution (0.5ug) immunoprecipitating Ku70 in HeLa whole cell lysate.

Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab92450 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab92450 in HeLa whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Ku70 antibody [EPR4027] (ab92450)

Predicted band size: 70 kDa

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• WB

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Western blot - Anti-Ku70 antibody [EPR4027] (AB92450)

All lanes:

Western blot - Anti-Ku70 antibody [EPR4027] (ab92450) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

A549 cell lysate at 10 µg

Lane 4:

A431 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 70 kDa

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• WB

Lab

Western blot - Anti-Ku70 antibody [EPR4027] (AB92450)

All lanes:

Western blot - Anti-Ku70 antibody [EPR4027] (ab92450) at 1/5000 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma 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: 70 kDa

Observed band size: 70 kDa

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• WB

CiteAb

Western blot - Anti-Ku70 antibody [EPR4027] (AB92450)

Ku70 western blot using anti-Ku70 antibody [EPR4027] ab92450. Publication image and figure legend from Xu, S., Kim, J., et al., 2020, Protein Cell, PubMed 32170574.

ab92450 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 ab92450 please see the product overview.

CAS9 interacts with KU86 and disrupts the formation of DNA-PK. (A and B) Reciprocal immunoprecipitation shows the interaction between CAS9 and KU86. Protein extracts from hESCs expressing CAS9 were immunoprecipitated with anti-KU86 (A) or anti-CAS9 (B), and immune precipitates were analyzed for the presence of KU86 and CAS9. (C) The interaction between CAS9 and KU86 was confirmed by the proximity ligation analysis (PLA). Cell nucleus were revealed by DAPI (Blue) staining and the CAS9-KU86 interaction indicated by red color. Scale bar, 25 μm. Unpaired t test. n = 20. Data are presented as mean value ± SD. ***p < 0.001. (D) Mapping the domain of CAS9 involved in the interaction with KU86. The Flag-tagged deletional mutants of CAS9 expressed in 293FT cells were immunoprecipitated with anti-flag antibody. Immune precipitates were analyzed for the presence of CAS9 mutants and KU86. (E) The expression of the PAM domain (1100) of CAS9 disrupted the interaction between CAS9 and KU86. The levels of CAS9, KU86, PAM in the input and immunoprecipitate were analyzed by Western blot. The ratio of CAS9 versus KU86 in the immunoprecipitate is shown at the bottom. (F) CAS9 disrupts the formation of DNA-PK complex. Protein extracts of cells in the presence and absence of CAS9 and DOX treatment were immunoprecipitated with anti-KU86 antibody. The levels of KU70, DNA-PKcs and CAS9 in the immunoprecipitate were analyzed. The relative ratios of DNA-PKcs versus KU86 or KU70 versus KU86 are indicated

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