Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue sections labeling RENT1/hUPF1 with Purified ab109363 at 1:100 dilution (5.14 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

ab109363 (purified) at 1:30 dilution (2µg) immunoprecipitating RENT1/hUPF1 in Raji whole cell lysate.
Lane 1 (input): Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysate 10µg
Lane 2 (+): ab109363 & Raji whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109363 in Raji whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling RENT1/hUPF1 with Purified ab109363 at 1:100 dilution (5.1 µg/ml). Cells were fixed in 100% Methanol and permeabilized with None. 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 nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling RENT1/hUPF1 with Purified ab109363 at 1:50 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. 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-RENT1/hUPF1 antibody [EPR4681] (ab109363) at 1/10000 dilution (Purified)

Lane 1 : HuT-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 124 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

All lanes : Anti-RENT1/hUPF1 antibody [EPR4681] (ab109363) at 1/10000 dilution

Lane 1 : HuT-78 cell lysate
Lane 2 : Raji cell lysate
Lane 3 : SH-SY5Y cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Performed under reducing conditions.

Predicted band size: 124 kDa

This image was generated using the unpurified version of the product. 

 

ab109363, at 1/100, staining RENT1/hUPF1 in Human kidney tissue by immunohistochemistry.

This image was generated using the unpurified version of the product. 

 

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling RENT1/hUPF1 with purified ab109363 at 1/500. Cells were fixed with 100% methanol. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control

This image was generated using the unpurified version of the product. 

 

ab109363, at 1/100, staining RENT1/hUPF1 in Human transitional cell carcinoma tissue by immunohistochemistry.

This image was generated using the unpurified version of the product. 

 

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

ab109363, at 1/100, staining RENT1/hUPF1 in HeLa cells by immunofluorescence.

This image was generated using the unpurified version of the product. 

 

Overlay histogram showing HeLa cells stained with ab109363 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab109363, 1/100 dilution) 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. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This image was generated using the unpurified version of the product.