All lanes : Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : RRM2B knockout HeLa cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 40 kDa

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.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling p53R2 with purified ab154194 at 1/50 dilution (2.2 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

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.

Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling p53R2 with purified ab154194 at 1/20 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : RRM2B knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 40 kDa
Observed band size: 40 kDa

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 : Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution (Purified)

Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Human skeletal muscle lysates
Lane 3 : SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

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 why is the actual band size different from the predicted?

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 : Anti-p53R2 antibody [EPR8816] (ab154194) at 1/1000 dilution ((unpurified))

Lane 1 : Human fetal muscle lysate
Lane 2 : MCF7 cell lysate
Lane 3 : SW480 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 40 kDa

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.

Immunofluorescent staining of MCF7 cells labeling p53R2 with unpurified ab154194 at 1/250 dilution.

Flow cytometric analysis of permeabilized MCF7 cells labeling p53R2 with unpurified ab154194 at 1/10 dilution (red) or a rabbit IgG negative control antibody (green).

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.

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.

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.

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.

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.

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.