All lanes : Anti-PCNA antibody [EPR3821] (ab92552) at 1/1000 dilution (purified)

Lane 1 : Mouse spleen lysate
Lane 2 : NIH/3T3 (Mouse embryo fibroblast cell line) lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded rat liver with purified ab92552 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded mouse testis with purified ab92552 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab92552 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Unpurified ab92552 showing positive staining in human normal colon tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Unpurified ab92552 showing positive staining in human urinary bladder carcinoma tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Unpurified ab92552 showing positive staining in human ovarian carcinoma tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Unpurified ab92552 showing positive staining in human breast carcinoma tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

Unpurified ab92552 (1/200) staining PCNA in Hela cells (green). Cells were fixed in methanol and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to abreview.

See Abreview

Immunofluorescence staining of A431 cells with purified ab92552 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab92552 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Unpurified ab92552 at 1/100 dilution staining PCNA in HeLa cells by Immunofluorescence.

All lanes : Anti-PCNA antibody [EPR3821] (ab92552) at 1/5000 dilution (purified)

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 29 kDa
Observed band size: 29 kDa

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

All lanes : Anti-PCNA antibody [EPR3821] (ab92552) at 1/50000 dilution (unpurified)

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate
Lane 3 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
Lane 4 : A431 (Human epidermoid carcinoma cell line) cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 29 kDa

ab92552 (purified) at 1/20 immunoprecipitating PCNA in 10 μg HeLa (Lanes 1 and 2, observed at 29 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

Overlay histogram showing HeLa cells fixed in 4% PFA and stained with purified ab92552 at a dilution of 1 in 40 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Overlay histogram showing HeLa cells stained with unpurified ab92552 (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 (ab92552, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.