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.

See Abreview

ICC/IF image of ab22604 stained human HepG2 cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab22604, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

All lanes : Anti-Glutathione Peroxidase 1 antibody (ab22604) at 1 µg/ml (Blocked with 3% Milk)

Lane 1 : Liver (Human) Tissue Lysate
Lane 2 : Liver (Mouse) Tissue Lysate
Lane 3 : Liver (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 22 kDa
Observed band size: 22 kDa
Additional bands at: 190 kDa, 55 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

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.

All lanes : Anti-Glutathione Peroxidase 1 antibody (ab22604) at 1 µg/ml

Lane 1 : Human liver normal tissue lysate
Lane 2 : Liver (Mouse) Tissue Lysate (ab7935)
Lane 3 : Human liver normal tissue lysate with Human Glutathione Peroxidase 1 peptide (ab25301) at 1 µg/ml
Lane 4 : Liver (Mouse) Tissue Lysate (ab7935) with Human Glutathione Peroxidase 1 peptide (ab25301) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat polyclonal to Rabbit IgG at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 22 kDa
Observed band size: 22,24 kDa why is the actual band size different from the predicted?

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).