Anti-Rad51 antibody [EPR4030(3)]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Immunofluorescent analysis of 100% Methanol-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling RAD51 with ab133534 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in HeLa cell line (shown in green).

The counterstain was observed in magenta.

Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (Magenta).

Secondary antibody only control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Immunofluorescent analysis of 4% PFA-fixed 0.1% Triton X-100 permeabilized UV-treated HCT116 cells labelling Rad51 with ab133534 at 0.2 μg/ml (shown in green). ab303656 Anti-gamma H2A.X (phospho S139) antibody [N1-431] was used as a DNA-damage counterstain at 0.2 μg/ml (shown in red). The secondary antibodies used were ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed and ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed, both were used at 1/1000 (2 μg/ml) dilution. Image shows selected areas of overlapping foci. The nuclear counterstain was DAPI.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Immunohistochemical analysis of formalin fixed paraffin embedded human testis labelling Rad51 with ab133534 at a concentration of 1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab133534 Anti-Rad51 antibody [EPR4030(3)] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

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 Fluorr^® 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Immunofluorescent analysis of 100% Methanol-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling RAD51 with ab133534 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in NIH/3T3 cell line (shown in green).

The counterstain was observed in magenta.

Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (Magenta).

Secondary antibody only control : Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) at 1/1000 dilution.

• WB

Lab

Western blot - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/10000 dilution

All lanes:

K562 (human chronic myelogenous leukemia cell line from bone marrow ) 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 1/50000 dilution

Predicted band size: 36 kDa

Observed band size: 37 kDa

false

• WB

Lab

Western blot - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/20000 dilution

Lane 1:

HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Jurkat (human T cell leukemia cell line from peripheral blood) 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 1/50000 dilution

Predicted band size: 21 kDa,36 kDa

Observed band size: 22 kDa,37 kDa

false

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Intracellular 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.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

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. Published online 2018 Sep 25. doi : 10.1038/s41467-018-06407-7; PMID : 30254264

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

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

• IP

Lab

Immunoprecipitation - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

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.

All lanes:

Immunoprecipitation - Anti-Rad51 antibody [EPR4030(3)] (ab133534)

Predicted band size: 36 kDa

Observed band size: 37 kDa

false

• WB

Lab

Western blot - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/10000 dilution

Lane 1:

C6 (rat glial tumor cell line) whole cell lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryo fibroblast cell line) 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 1/50000 dilution

Predicted band size: 36 kDa

Observed band size: 37 kDa

false

• WB

Lab

Western blot - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

Different batches of ab133534 were tested on HEK-293 (Human embryonic kidney epithelial cell) lysate at 2.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 37 kDa.

All lanes:

Western blot - Anti-Rad51 antibody [EPR4030(3)] (ab133534)

Predicted band size: 36 kDa

false

• WB

Unknown

Western blot - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

All lanes:

Western blot - Anti-Rad51 antibody [EPR4030(3)] (ab133534) at 1/10000 dilution

Lane 1:

C6 (rat glial tumor cell line) cell lysate at 10 µg

Lane 2:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate at 10 µg

Lane 3:

Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate at 10 µg

Lane 4:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Lane 5:

K562 (human chronic myelogenous leukemia cell line from bone marrow ) cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 36 kDa

Observed band size: 37 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Rad51 antibody [EPR4030(3)] (AB133534)

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.