Recombinant Anti-Glutathione Peroxidase 1 antibody [EPR3311] (ab108429)

False colour image of Western blot: Anti-Glutathione Peroxidase 1 antibody [EPR3311] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab108429 was shown to bind specifically to Glutathione Peroxidase 1. A band was observed at 22 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in GPX1 knockout cell line ab266650 (knockout cell lysate ab256932). To generate this image, wild-type and GPX1 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: GPX1 knockout HAP1 cell lysate (20 µg)
Lane 3: THP1 cell lysate (20 µg)
Lane 4: HL60 cell lysate (20 µg)
Lanes 1 and 2: Merged signal (red and green). Green - ab108429, observed at 22 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab108429 was shown to specifically react with Glutathione Peroxidase 1 in wild-type HAP1 cells. No band was observed when Glutathione Peroxidase 1 knockout samples were examined. Wild-type and Glutathione Peroxidase 1 knockout samples were subjected to SDS-PAGE. ab108429 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively 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 hour at room temperature before imaging.

Intracellular Flow Cytometry analysis of THP-1 (human acute monocytic leukemia) cells labeling Glutathione Peroxidase 1 with purified ab108429 at 1/250 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) (ab172730) was used as the isotype control, Cell without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

ab108429, at 1/100 dilution, staining Glutathione Peroxidase 1 in paraffin-embedded Human breast carcinoma tissue by Immunohistochemistry.

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