GPX4 Recombinant Rabbit Monoclonal Antibody [JU11-31]
cat.: ET1706-45
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat, Zebrafish
Applications: WB, IHC-P, IF-Cell, FC
Clonality: Monoclonal
Clone number: JU11-31
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 22 kDa
Isotype: IgG
Immunogen: Synthetic peptide within Human GPX4 aa 23-72 / 197.
Positive control: HeLa cell lysate, A549 cell lysate, HepG2 cell lysate, U-937 cell lysate, HEK-293 cell lysate, mouse testis tissue lysate, rat testis tissue lysate, mouse kidney tissue lysate, rat kidney tissue lysate, mouse brain tissue lysate, rat brain tissue lysate, zebrafish tissue lysates, HEK-293, rat epididymis tissue, human kidney tissue, mouse testis tissue, MCF-7, PC-3M, LOVO.
Subcellular location: Mitochondrion, Cytoplasm.
Recommended Dilutions:
  WB
  IHC-P
  IF-Cell
  FC
1:10,000
1:200-1:1,000
1:50-1:200
1: 10,000
Uniprot #: SwissProt: P36969 Human | O70325 Mouse | P36970 Rat
Alternative names: Glutathione peroxidase 4 GPX 4 GPX-4 GPX4 GPX4_HUMAN GSHPx-4 MCSP mitochondrial PHGPx Phospholipid hydroperoxidase Phospholipid hydroperoxide glutathione peroxidase Phospholipid hydroperoxide glutathione peroxidase mitochondrial snGPx snPHGPx Sperm nucleus glutathione peroxidase
Images
ET1706-45_1.jpg Fig1: Immunocytochemistry analysis of HEK-293 cells labeling GPX4 with Rabbit anti-GPX4 antibody (ET1706-45) at 1/200 dilution and competitor's antibody at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-GPX4 antibody (ET1706-45) at 1/200 dilution and competitor's antibody at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1706-45_2.jpg Fig2: Western blot analysis of GPX4 on different lysates with Rabbit anti-GPX4 antibody (ET1706-45) at 1/10,000 dilution and competitor's antibody at 1/10,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: A549 cell lysate (20 µg/Lane)
Lane 3: HepG2 cell lysate (20 µg/Lane)
Lane 4: U-937 cell lysate (20 µg/Lane)
Lane 5: HEK-293 cell lysate (20 µg/Lane)
Lane 6: Mouse testis tissue lysate (20 µg/Lane)
Lane 7: Rat testis tissue lysate (20 µg/Lane)
Lane 8: Mouse kidney tissue lysate (20 µg/Lane)
Lane 9: Rat kidney tissue lysate (20 µg/Lane)
Lane 10: Mouse brain tissue lysate (20 µg/Lane)
Lane 11: Rat brain tissue lysate (20 µg/Lane)

Predicted band size: 22 kDa
Observed band size: 20 kDa

Exposure time: 2 minutes;
4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1706-45) at 1/10,000 dilution and competitor's antibody at 1/10,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.
ET1706-45_3.jpg Fig3: Western blot analysis of GPX4 on zebrafish tissue lysates with Rabbit anti-GPX4 antibody (ET1706-45) at 1/500 dilution.

Lysates/proteins at 10 µg/Lane.

Predicted band size: 22 kDa
Observed band size: 17 kDa

Exposure time: 2 minutes;

12% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1706-45) at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:5,000 dilution was used for 1 hour at room temperature.
ET1706-45_4.jpg Fig4: Western blot analysis of GPX4 on different lysates with Rabbit anti-GPX4 antibody (ET1706-45) at 1/500 dilution.

Lane 1: Hela-si NT cell lysate
Lane 2: Hela-si GPX4 cell lysate

Lysates/proteins at 10 µg/Lane.

Predicted band size: 22 kDa
Observed band size: 20,22 kDa

Exposure time: 1 minutes 40 seconds;

4-20% SDS-PAGE gel.

ET1706-45 was shown to specifically react with GPX4 in Hela-si NT cells. Weakened band was observed when Hela-si GPX4 sample was tested. Hela-si NT and Hela-si GPX4 samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (ET1706-45, 1/500) and Loading control antibody (Rabbit anti-GAPDH , ET1601-4, 1/10,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-rabbit IgG-HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
ET1706-45_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded mouse testis tissue with Rabbit anti-GPX4 antibody (ET1706-45) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1706-45) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
ET1706-45_6.jpg Fig6: Immunocytochemistry analysis of MCF-7 cells labeling GPX4 with Rabbit anti-GPX4 antibody (ET1706-45) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-GPX4 antibody (ET1706-45) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
ET1706-45_7.jpg Fig7: Immunocytochemistry analysis of PC-3M cells labeling GPX4 with Rabbit anti-GPX4 antibody (ET1706-45) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-GPX4 antibody (ET1706-45) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
ET1706-45_8.jpg Fig8: Immunocytochemistry analysis of LOVO cells labeling GPX4 with Rabbit anti-GPX4 antibody (ET1706-45) at 1/50 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-GPX4 antibody (ET1706-45) at 1/50 dilution in 2% negative goat serum overnight at 4 ℃. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
ET1706-45_9.jpg Fig9: Immunocytochemistry analysis of HEK-293 cells labeling GPX4 with Rabbit anti-GPX4 antibody (ET1706-45) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-GPX4 antibody (ET1706-45) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1706-45_10.jpg Fig10: Immunocytochemistry analysis of C6 cells labeling GPX4 with Rabbit anti-GPX4 antibody (ET1706-45) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-GPX4 antibody (ET1706-45) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
ET1706-45_11.jpg Fig11: Flow cytometric analysis of HEK-293 cells labeling GPX4.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1706-45, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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