Recombinant Anti-RPA70 antibody [EPR3472] (ab79398)

Blocking/Dilution buffer: 5% NFDM/TBST.

Unpurified ab79398 staining RPA70 in U-2 OS (Human bone osteosarcoma epithelial cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS and blocked with 2% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/500 in PBS + 0.5% Tween-20) for 2 hours at 25°C. A Cy3^®-conjugated goat anti-rabbit IgG monoclonal (1/250) was used as the secondary antibody.

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Immunocytochemistry/Immunofluorescence analysis of A549 (Human lung carcinoma cell line) cells labeling RPA70 with purified ab79398 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 (1/500) 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 (1/500) were also used.

Control 1: primary antibody (1/200) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labeling RPA70 with purified ab79398 at 1/100.

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.

ab79398 (purified) at 1/20 immunoprecipitating RPA70 in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate. 

Lane 1 (input): HeLa whole cell lysate (10 µg)

Lane 2 (+): ab79398 + HeLa whole cell lysate (10 µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab79398 in HeLa whole cell lysate.

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling RPA70 with purified ab79398 at 1/80 (red). 

Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies. 

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical squamous cell carcinoma labelling RPA70 with unpurified ab79398 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 unpurifiedab79398 (red line). 

The cells were fixed with 80% methanol (5 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 (unpurified ab79398, 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.