Anti-Glutathione Peroxidase 1 antibody (ab22604)

ab22604 staining Glutathione Peroxidase 1 in HepG2 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab22604 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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 - 4: Merged signal (red and green). Green - ab22604 observed at 24 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab22604 was shown to recognize Glutathione Peroxidase 1 in wild-type HAP1 cells along with additional cross-reactive bands. 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. ab22604 at a concentration of 1µg/ml and ab8245 (loading control to GAPDH) diluted at 1/2000 were 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.

ab22604 staining mouse Cor-1 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with TBS/BSA/azide/0.1%Tween 20 and blocked with 1% BSA for 10 minutes at RT. Samples were incubated with primary antibody (1/300) for 2 hours. An diluted (1/1000) Alexa Fluor® 594-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab22604 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

ab22604 detects a band of approximately 22 kDa in human liver lysate. The band detected in the mouse liver lysate runs slightly higher (~ 24kDa). We believe that these bands correspond to Glutathione Peroxidase 1 as they are both blocked by the addition of the immunizing peptide (ab25301).