Anti-PABPN1 antibody [EP3000Y] (ab75855) at 1/2000 dilution (purified) + Rat brain lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 33 kDa

Blocking and diluting buffer: 5% NFDM/TBST

Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PABPN1 with purified ab75855 at 1:40 dilution (10 ug/ml) (red). Cells were fixed with 80% Methanol and permeabilized with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor^®488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PABPN1 with purified ab75855 at 1:100 dilution (4.1μg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ab75855 (purified) at 1:30 dilution (5ug) immunoprecipitating PABPN1 in MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate.
Lane 1 (input): MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab75855 & MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab75855 in MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:10,000 dilution.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling PABPN1 with Purified ab75855 at 1:1000 dilution (0.41 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling PABPN1 with Purified ab75855 at 1:1000 dilution (0.41 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue sections labeling PABPN1 with Purified ab75855 at 1:1000 dilution (0.41 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling PABPN1 with unpurified ab75855 at 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI.

All lanes : Anti-PABPN1 antibody [EP3000Y] (ab75855) at 1/2000 dilution (purified)

Lane 1 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 2 : Mouse spleen lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa

Blocking and diluting buffer: 5% NFDM/TBST

All lanes : Anti-PABPN1 antibody [EP3000Y] (ab75855) at 1/2000 dilution (purified)

Lane 1 : 293T (Human embryonic kidney epithelial cell) whole cell lysates
Lane 2 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 33 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

Lanes 1-2 : Anti-PABPN1 antibody [EP3000Y] (ab75855) at 1/200000 dilution (unpurified)
Lane 3 : Anti-PABPN1 antibody [EP3000Y] (ab75855) at 1/1000000 dilution (unpurified)

Lane 1 : Raw264.7 cell lysate
Lane 2 : MCF-7 cell lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/1000 dilution

Predicted band size: 33 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?

Overlay histogram showing MCF-7 cells stained with unpurified ab75855 (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 (ab75855, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Unpurified ab75855, at 1/100 dilution, staining PABPN1 in squamous cell cervical carcinoma, by Immunohistochemistry using formalin-fixed, paraffin-embedded tissue.

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