Immunocytochemistry/Immunofluorescence analysis of Jurkat (human T cell leukemia cell line from peripheral blood) cells labelling Rad51 with purified ab133534 at 1/1000. 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 (1/1000) 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).

All lanes : Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/10000 dilution (purified)

Lane 1 : C6 (rat glial tumor cell line) whole cell lysate
Lane 2 : Mouse spleen tissue lysate
Lane 3 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 37 kDa
Observed band size: 37 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labelling Rad51 with purified ab133534 at 1/500. 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.

Flow Cytometry analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling Rad51 with purified ab133534 at 1/350 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab133534 (purified) at 1/100 immunoprecipitating Rad51 in HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate.

Lane 1 (input): HEK-293 whole cell lysate (10µg)

Lane 2 (+): ab133534 + HEK-293 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab133534 in HEK-293 whole cell lysate.

For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Immunocytochemical analysis of DT40 cells (WT and the indicated KOs) labeling Rad51 using ab133534 at 1/200 dilution.

From Figure 5d of gao et al.

Gao et al Nat Commun. 2018; 9: 3925. Published online 2018 Sep 25. doi: 10.1038/s41467-018-06407-7

Reproduced under the Creative Commons Licence http://creativecommons.org/licenses/by/4.0/.

Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/10000 dilution (purified) + K562 (human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 37 kDa
Observed band size: 37 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/20000 dilution (purified)

Lane 1 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 37 kDa
Observed band size: 37 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/10000 dilution (unpurified)

Lane 1 : C6 (rat glial tumor cell line) cell lysate
Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
Lane 3 : Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate
Lane 4 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 5 : K562 (human chronic myelogenous leukemia cell line from bone marrow ) cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 37 kDa
Observed band size: 37 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue labelling Rad51 with unpurified ab133534 at a dilution of 1/100.

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

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with unpurified ab133534 (red line). The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (unpurified ab133534, 1/1000 dilution) for 30 minutes at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1 μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Equilibrium disassociation constant (K_D)
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