Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling 53BP1 with purified ab175933 at 1/200. 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 (1/200) 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).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: 53BP1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (40 µg)
Lane 4: HepG2 cell lysate (40 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab175933 observed at 350 kDa. Red - loading control, ab18058, observed at 124 kDa.
ab175933 was shown to specifically react with 53BP1 when 53BP1 knockout samples were used. Wild-type and 53BP1 knockout samples were subjected to SDS-PAGE. ab175933 and ab18058 (loading control to Vinculin) were diluted 1/1000 and 1/10000 respectively 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 1/10000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling 53BP1 with purified ab175933 at a dilution of 1/60. 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.

ab175933 staining 53BP1 in the human cell line HepG2 (human hepatocellular carcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/30. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: 53BP1 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (40 µg)
Lane 4: HepG2 cell lysate (40 µg)

Lanes 1 - 4: Merged signal (red and green).

Green - Target observed at 350 kDa. Red - loading control, ab18058, observed at 124 kDa.

This western blot image is a comparison between ab175933 and a competitor's top cited rabbit polyclonal antibody.

All lanes : Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 1/1000 dilution (purified)

Lane 1 : Mouse heart tissue lysate
Lane 2 : Mouse brain tissue lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 214 kDa
Observed band size: 450 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM /TBST.

Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 20 µg (purified) + Rat heart tissue lysate at 20 µg

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

Predicted band size: 214 kDa
Observed band size: 450 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling 53BP1 with purified ab175933 at a dilution of 1/60. 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.

All lanes : Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 1/5000 dilution (purified)

Lane 1 : Human fetal heart tissue lysate
Lane 2 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 214 kDa
Observed band size: 450 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM /TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver carcinoma tissue labelling 53BP1 with purified ab175933 at a dilution of 1/60. 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.

All lanes : Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 1/1000 dilution (unpurified)

Lane 1 : HepG2 cell lysate
Lane 2 : Human fetal brain lysate
Lane 3 : Human fetal heart lysate

Lysates/proteins at 1/10 dilution per lane.

Predicted band size: 214 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling 53BP1 with unpurified ab175933 at a dilution of 1/100.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling 53BP1 with unpurified ab175933 at a dilution of 1/100.

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

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling 53BP1 with unpurified ab175933 at a dilution of 1/100.