Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free

• ICC/IF

AbReview45357****

Immunocytochemistry/ Immunofluorescence - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab125066).

This image is courtesy of an Abreview submitted by Kirk McManus

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

This data was developed using the same antibody clone in a different buffer formulation (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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

This data was developed using the same antibody clone in a different buffer formulation (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab125066).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab125066).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab125066).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab125066).

• WB

Lab

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] - BSA and Azide free (AB219592)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/glutathione-peroxidase-4-antibody-epncir144-ab125066'>ab125066</a>) 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='/en-us/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] - BSA and Azide free (AB219592)

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. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab125066).

All lanes:

Western blot - Anti-Glutathione Peroxidase 4 antibody [EPNCIR144] (<a href='/en-us/products/primary-antibodies/glutathione-peroxidase-4-antibody-epncir144-ab125066'>ab125066</a>) 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] - BSA and Azide free (AB219592)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/glutathione-peroxidase-4-antibody-epncir144-ab125066'>ab125066</a>) 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='/en-us/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