Recombinant Anti-Peroxiredoxin 2/PRP antibody [EPR5154] (ab109367)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Peroxiredoxin 2/PRP knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: LnCaP cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab109367 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109367 was shown to specifically react with Peroxiredoxin 2/PRP when Peroxiredoxin 2/PRP knockout samples were used. Wild-type and Peroxiredoxin 2 knockout samples were subjected to SDS-PAGE. ab109367 and ab8245 (loading control to GAPDH) 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 1/10 000 dilution for 1 h at room temperature before imaging.

Immunofluorescence staining of HeLa cells with purified ab109367 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 100% methanol 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 ab109367 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.

Lanes 1- 2: Merged signal (red and green). Green - ab109367 observed at 22 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109367 was shown to react with Peroxiredoxin 2/PRP in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266392 (knockout cell lysate ab257041) was used. Wild-type HEK-293T and PRDX2 knockout HEK-293T 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. ab109367 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.

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

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

Overlay histogram showing HeLa cells fixed in 80% methanol and stained with purified ab109367 at a dilution of 1/200 (red line). The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit at a dilution of 1/500. 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 unpurifiedab109367 (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 (ab109367, 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.

Immunofluorescent staining of Peroxiredoxin 2/PRP in HeLa cells using unpurified ab109367 at 1/250 dilution.

Immunohistochemical analysis of Peroxiredoxin 2/PRP in paraffin-embedded Human prostatic hyperplasia tissue using unpurified ab109367 at 1/1000 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of Peroxiredoxin 2/PRP in paraffin-embedded Human stomach tissue using unpurified ab109367 at 1/1000 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Unpurified ab109367 (1/500) staining Peroxiredoxin 2/PRP in asynchronous 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