Recombinant Anti-PCNA antibody [EPR3821] (ab92552)

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

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.

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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.

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

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 Fluorr^® 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.