All lanes : Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : PRDX1 knockout HEK293T cell lysate
Lane 3 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 22 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab109498 observed at 26 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab109498 Anti-Peroxiredoxin 1/PAG antibody [EPR5433] was shown to specifically react with Peroxiredoxin 1/PAG in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266842 (knockout cell lysate ab257040) was used. Wild-type and Peroxiredoxin 1/PAG knockout samples were subjected to SDS-PAGE. ab109498 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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.

Immunohistochemical analysis of paraffin-embedded human kidney tissue using ab109498 at 1/250

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

Overlay histogram showing HeLa cells stained with ab109498 (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 (ab109498, 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) (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 analysis of 293T cells labeled with ab109498 at 1/10.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Peroxiredoxin 1/PAG knockout HAP1 cell lysate (20 µg)
Lane 3: A431 cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab109498 observed at 23 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109498 was shown to specifically react with Peroxiredoxin 1/PAG when Peroxiredoxin 1/PAG knockout samples were used. Wild-type and Peroxiredoxin 1/PAG knockout samples were subjected to SDS-PAGE. ab109498 and ab8245 (loading control to GAPDH) were both diluted 1/10 000 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.

All lanes : Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) at 1/10000 dilution

Lane 1 : MCF-7 cell lysate
Lane 2 : 293T cell lysate
Lane 3 : K562 cell lysates

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 22 kDa
Observed band size: 22 kDa

Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue using ab109498 at 1/250.

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