Anti-p53R2 antibody [EPR8816]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemical analysis of paraffin-embedded Wild-type HeLa (Human cervical adenocarcinoma epithelial cell) cell pellet (A) and RRM2B KO HeLa cell pellet (B) labelling p53R2 with ab154194 at 1 : 2500 (0.044 μg/ml). Positive staining on (A) wild-type HeLa cell pellet, no staining on (B) RRM2B KO HeLa A431 cell pellet. The primary antibody was incubated for 30 mins at room temperature. Secondary antibody was a Ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemical analysis of paraffin embedded Human normal colon tissue using unpurified ab154194 showing +ve staining.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemical analysis of paraffin embedded Human normal kidney tissue unpurified ab154194 showing +ve staining.

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-p53R2 antibody [EPR8816] (AB154194)

Intracellular flow cytometric analysis of permeabilized MCF7 cells labeling p53R2 with unpurifiedab154194 at 1/10 dilution (red) or a rabbit IgG negative control antibody (green).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemical analysis of paraffin-embedded Human ovarian carcinoma tissue labeling p53R2 with unpurified ab154194 at 1/50 dilution.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemical analysis of paraffin embedded Human melanoma tissue using unpurified ab154194 showing +ve staining.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemical analysis of paraffin embedded Human urinary bladder transitional carcinoma tissue using unpurified ab154194 showing +ve staining.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemical analysis of paraffin embedded Human normal brain tissue using unpurified ab154194 showing +ve staining.

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p53R2 antibody [EPR8816] (AB154194)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling p53R2 with purified ab154194 at 1/500 dilution (0.22 μg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody.

Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

Unknown

Immunoprecipitation - Anti-p53R2 antibody [EPR8816] (AB154194)

ab154194 (purified ) at 1/20 dilution (0.5ug) immunoprecipitating p53R2 in MCF7 whole cell lysate.

Lane 1 (input) : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab154194 & MCF7 whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab154194 in MCF7 whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-p53R2 antibody [EPR8816] (ab154194)

Predicted band size: 40 kDa

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• WB

Lab

Western blot - Anti-p53R2 antibody [EPR8816] (AB154194)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : p53R2 knockout HAP1 cell lysate (20 μg)
Lane 3 : MCF7 cell lysate (20 μg)
Lane 4 : SW480 cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab154194 observed at 40 kDa. Red - loading control, ab18058, observed at 124 kDa.

Unpurified ab154194 was shown to recognize p53R2 when p53R2 knockout samples were used, along with additional cross-reactive bands. Wild-type and p53R2 knockout samples were subjected to SDS-PAGE. ab154194 and ab18058 (loading control to Vinculin) were both diluted 1/1000 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 h at room temperature before imaging.

All lanes:

Western blot - Anti-p53R2 antibody [EPR8816] (ab154194)

Predicted band size: 40 kDa

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• WB

Lab

Western blot - Anti-p53R2 antibody [EPR8816] (AB154194)

Lanes 1- 2 : Merged signal (red and green). Green - ab154194 observed at 40 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab154194 was shown to react with p53R2 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261769 (knockout cell lysate ab257215) was used. Wild-type HeLa and RRM2B knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab154194 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 40 µg

Lane 2:

RRM2B knockout HeLa cell lysate at 40 µg

Lane 2:

Western blot - Human RRM2B (p53R2) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rrm2b-p53r2-knockout-hela-cell-line-ab261769'>ab261769</a>)

Predicted band size: 40 kDa

Observed band size: 40 kDa

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• WB

Lab

Western blot - Anti-p53R2 antibody [EPR8816] (AB154194)

Lanes 1- 2 : Merged signal (red and green). Green - ab154194 observed at 40 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab154194 was shown to react with p53R2 in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266897 (knockout cell lysate ab257216) was used. Wild-Type HCT116 and RRM2B knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab154194 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution

Lane 1:

Wild-type HCT116 cell lysate at 20 µg

Lane 2:

RRM2B knockout HCT116 cell lysate at 20 µg

Lane 2:

Western blot - Human RRM2B (p53R2) knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-rrm2b-p53r2-knockout-hct116-cell-line-ab266897'>ab266897</a>)

Predicted band size: 40 kDa

Observed band size: 40 kDa

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• WB

Unknown

Western blot - Anti-p53R2 antibody [EPR8816] (AB154194)

All lanes:

Western blot - Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution

Lane 1:

Human fetal muscle lysate at 10 µg

Lane 2:

MCF7 cell lysate at 10 µg

Lane 3:

SW480 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 130 kDa,40 kDa

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• WB

Lab

Western blot - Anti-p53R2 antibody [EPR8816] (AB154194)

All lanes:

Western blot - Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

Human skeletal muscle lysates at 20 µg

Lane 3:

SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 40 kDa

Observed band size: 41 kDa

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