Anti-Glutathione Peroxidase 4 抗体 [EPNCIR144]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Immunohistochemical analysis of formalin fixed paraffin embedded human testis labelling Glutathione Peroxidase 4 with ab125066 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab125066 anti-Glutathione Peroxidase 4 antibody [EPNCIR144] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Glutathione Peroxidase 4 (red) with ab125066 at a 1/400 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluorr^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Unpurified ab125066 at a 1/100 dilution staining Glutathione Peroxidase 4 in paraffin-embedded human kidney tissue by immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

AbReview45357****

Immunocytochemistry/ Immunofluorescence - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Unpurified ab125066 staining Glutathione Peroxidase 4 in the HeLa cell line from Human cervical cancer by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with methanol. Samples were incubated with primary antibody (1/500 dilution in PBS) for 1 hour at 22°C. ab150081 an Alexa Fluor^® 488-conjugated Goat anti-rabbit IgG polyclonal (1/200 dilution) was used as the secondary antibody. Nuclear staining was carried out with DAPI.

This image is courtesy of an Abreview submitted by Kirk McManus

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Immunofluorescence staining of HEK293 cells with purified ab125066 at a working dilution of 1/200 counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077) used at a dilution of 1/1000. ab7291 a mouse anti-tubulin antibody (1/1000) was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse 1/1000) shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1 purified ab125066 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2 ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Immunohistochemical staining of paraffin embedded human stomach with purified ab125066 at a working dilution of 1/50. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500 dilution. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Immunohistochemical analysis of formalin fixed paraffin embedded human testis labelling Glutathione Peroxidase 4with ab125066 at a concentration of 1 µg/ml. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab125066 anti-Glutathione Peroxidase 4 antibody [EPNCIR144] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

• WB

Lab

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Blocking buffer and concentration : 5% NFDM/TBST  Diluting buffer and concentration : 5% NFDM/TBST This antibody can detect 19kda cytoplasmic form and 22kda mitochondrial isoform.

All lanes:

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (ab125066) at 1/1000 dilution

Lane 1:

Rat testis tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 22 kDa

Observed band size: 19 kDa,22 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Lanes 1 - 3 : Merged signal (red and green). Green - ab125066 observed at 20 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab125066 was shown to react with Glutathione Peroxidase 4 in wild-type HeLa cells in Western blot with loss of signal observed in GPX4 knockout cell line ab262509 (knockout cell lysate ab263935). Wild-type HeLa and GPX4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab125066 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (ab125066) at 1/1000 dilution

Lane 1:

Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

GPX4 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

Western blot - Human GPX4 knockout HeLa cell line (<a href='/products/cell-lines/human-gpx4-knockout-hela-cell-line-ab262509'>ab262509</a>)

Lane 3:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Predicted band size: 22 kDa

Observed band size: 20 kDa

false

• WB

Lab

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Blocking/Diluting buffer and concentration 5% NFDM/TBST. ab181602 was used as GAPDH loading control.

All lanes:

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (ab125066) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Rat heart lysate at 20 µg

Lane 5:

Mouse ovary lysate at 20 µg

Lane 6:

Mouse heart lysate at 20 µg

Lane 7:

Mouse lung lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Blocking buffer : 5% NFDM/TBST.
Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (ab125066) at 1/20000 dilution

Lane 1:

Mouse testis lysate at 20 µg

Lane 2:

Rat testis lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 22 kDa

Observed band size: 17 kDa

false

• Biochemical assay

PubMed

Biochemical assay - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (AB125066)

Niu et al used Lipid Peroxidation (MDA) Assay Kit ab118970, GSH/ GSSG Assay Kit ab138881, and Glutathione Peroxidase Assay Kit ab125066 to investigate the use of porous Se@SiO2 nanospheres to achieve controlled release of selenium, a scavenger of intracellular free radicals, in mouse eyes.

Male diabetic db/db and control db/m mice were injected in the eyes with porous Se@SiO2 nanospheres, and porous SiO2 nanospheres (NPs) without Se.

Porous Se@SiO2 nanospheres inhibit diabetes-induced retinal lipid peroxidation and inflammation. Levels of MDA in retinal homogenates (n = 6). Expression levels of GPX4 in retinas were measured using western blotting; β-actin was used as a loading control (left panel). Band densities were assessed using the ImageJ software, and GPX4 expression levels are represented as their ratios to β-actin (right panel) (n = 3). Levels of GSH, GSSG, and the ratio of GSH to GSSG in retinal homogenates (n = 6). Data are represented as the mean ± SD. **p < 0.01, ***p < 0.001; ns, nonsignificant.

Retinal samples were harvested, washed, and lysed according to the manufacturer's instructions. Protein concentrations were determined using a bicinchoninic acid kit. MDA and glutathione concentrations were determined using a lipid peroxidation MDA assay kit, reduced glutathione (GSH) / oxidized glutathione (GSSG) Ratio Detection Assay kit (ab118970, ab138881; Abcam, MA, USA), and a microplate reader. The relative concentrations of MDA and glutathione were calculated by normalizing the measured concentrations to that of the total protein.