Anti-RPA70 antibody [EPR3472]

• ICC/IF

AbReview46343****

Immunocytochemistry/ Immunofluorescence - Anti-RPA70 antibody [EPR3472] (AB79398)

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.

This image is courtesy of an Abreview submitted by Remi Buisson.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RPA70 antibody [EPR3472] (AB79398)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RPA70 antibody [EPR3472] (AB79398)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RPA70 antibody [EPR3472] (AB79398)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RPA70 antibody [EPR3472] (AB79398)

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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RPA70 antibody [EPR3472] (AB79398)

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.

• IP

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Immunoprecipitation - Anti-RPA70 antibody [EPR3472] (AB79398)

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.

All lanes:

Immunoprecipitation - Anti-RPA70 antibody [EPR3472] (ab79398)

Predicted band size: 68 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-RPA70 antibody [EPR3472] (AB79398)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-RPA70 antibody [EPR3472] (ab79398) at 1/1000 dilution

All lanes:

A549 (Human lung carcinoma cell line) cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 70 kDa

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• WB

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Western blot - Anti-RPA70 antibody [EPR3472] (AB79398)

All lanes:

Western blot - Anti-RPA70 antibody [EPR3472] (ab79398) at 1/5000 dilution

Lane 1:

A549 (Human lung carcinoma cell line) cell lysate at 10 µg

Lane 2:

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

Secondary

All lanes:

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

Predicted band size: 68 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-RPA70 antibody [EPR3472] (AB79398)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-RPA70 antibody [EPR3472] (ab79398) at 1/4000 dilution

Lane 1:

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

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 68 kDa

Observed band size: 70 kDa

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• WB

CiteAb

Western blot - Anti-RPA70 antibody [EPR3472] (AB79398)

RPA70 western blot using anti-RPA70 antibody [EPR3472] ab79398. Publication image and figure legend from Pedersen, H., Anne Adanma Obara, E., et al., 2020, Int J Mol Sci, PubMed 32111042.

ab79398 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab79398 please see the product overview.

RPA expression is crucial for the maintenance of glioblastoma cancer stem-like cells. (A) Immunoblot analysis of RPA70, RPA32 and RPA14 in a panel of GBM primary cell lines (4121, G01, G06, G40, G07, G16 and G20) and two normal human astrocyte cell lines (NHA33, NHA26). β-Actin was used as a loading control. (B) Immunoblot analysis of RPA70, RPA32 and RPA14 expression in matched pairs of glioblastoma cancer stem-like cells (GSCs) and differentiated GBM cells (DGCs) isolated from 4121, G01, G06 and G40 primary GBM cell lines. α-Tubulin or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) Immunoblot analysis validating knockdown efficiencies of lentiviral shRNAs targeting RPA70, RPA32 and RPA14 in G01-GSCs. α-Tubulin was used as a loading control. (D) CellTiter-Glo luminescent viability assay (Promega) of G01-GSCs transduced with lentiviral shRNA targeting RPA70, RPA32 or RPA14 assessed 72 h after virus wash. Data are presented as mean ± SD. Statistical significance was tested using a two-way ANOVA with post-hoc Sidak's multiple comparisons test, where *** p < 0.001. N = 3. (E) CellTiter-Glo luminescent viability assay (Promega) of G01-GSCs transduced with lentiviral shRNA targeting RPA70, RPA32 or RPA14 alone or 24 h after irradiation (3 Gy; COMBO) or sham-irradiated and assessed for viability 72 h later. (E) Extreme limiting dilution assay (ELDA) of G01-GSCs transduced with lentiviral shRNA targeting of RPA70 (2 independent shRNAs) shows significantly impaired self-renewal of G01-GSCs after 10 days. Data are presented as mean ± SD. Statistical significance was tested using a two-way ANOVA with post-hoc Sidak's multiple comparisons test, where * p < 0.05, ** p < 0.01, *** p < 0.001. N = 3.

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