All lanes : Anti-Ku70 antibody [EPR4027] (ab92450) at 1/5000 dilution (Purified)

Lane 1 : A549 (Human lung carcinoma epithelial cell) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 70 kDa
Observed band size: 70 kDa

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.

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.

All lanes : Anti-Ku70 antibody [EPR4027] (ab92450) at 1/1000 dilution ((unpurified))

Lane 1 : HeLa cell lysate
Lane 2 : 293T cell lysate
Lane 3 : A549 cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 70 kDa

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Ku70 with purified ab92450 at 1/20 dilution (5 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 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).

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.

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

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 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. 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.

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.