Anti-xCT antibody [EPR27115-64]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue labeling xCT with ab307601 at 1/500 dilution (1.024 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human thyroid carcinoma.

The section was incubated with ab307601 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labeling xCT with ab307601 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membranous and cytoplasmic staining in A549 cell line. Low expression : COLO 205 (PMID : 31000598). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/ml) (Red). Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling xCT with ab307601 at 1/500 dilution (1.024 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human cerebrum. The section was incubated with ab307601 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling xCT with ab307601 at 1/500 dilution (1.024 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on human skeletal muscle. The section was incubated with ab307601 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-xCT antibody [EPR27115-64] (AB307601)

Flow cytometric analysis of 4% paraformaldehyde-fixed 90% methanol-permeabilized COLO 205 (human colon adenocarcinoma tumor ascitic epithelial cell) (Left panel) / A549 (human lung carcinoma epithelial cell) (Right panel) cells labeling xCT with ab307601 at 1/50 dilution (1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Low expression : COLO 205 (PMID : 31000598).

• IP

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Immunoprecipitation - Anti-xCT antibody [EPR27115-64] (AB307601)

xCT was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate 10 µg with ab307601 at 1/30 dilution (2 µg in 0.3 5mg lysates). Western blot was performed on the immunoprecipitate using ab307601 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : A549 whole cell lysate 10 µg Lane 2 : ab307601 IP in A549 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307601 in A549 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 41 seconds.

All lanes:

Immunoprecipitation - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/30 dilution

All lanes:

A549 (human lung carcinoma epithelial cell)

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 37 kDa

false

Exposure time: 41s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling xCT with ab307601 at 1/500 dilution (1.024 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat cerebrum. The section was incubated with ab307601 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded (A) Cerebrum tissue from wild-type C57BL/6J mice and (B) Cerebrum tissue from xCT knockout mice tissue labeling xCT with ab307601 at 1/1000 dilution.

Positive staining on (A) Cerebrum tissue from wild-type C57BL/6J mice, no staining on (B) Cerebrum tissue from xCT knockout mice.

The primary antibody was incubated for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Slc7a11-KO homozygous mice (Strain ID : T015610).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded rat skeletal muscle tissue labeling xCT with ab307601 at 1/500 dilution (1.024 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on rat skeletal muscle (PMID : 17401668). The section was incubated with ab307601 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded mouse skeletal muscle tissue labeling xCT with ab307601 at 1/500 dilution (1.024 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on mouse skeletal muscle. The section was incubated with ab307601 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-xCT antibody [EPR27115-64] (AB307601)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling xCT with ab307601 at 1/500 dilution (1.024 µg/ml) followed by ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse cerebrum (PMID : 29350434). The section was incubated with ab307601 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• WB

Lab

Western blot - Anti-xCT antibody [EPR27115-64] (AB307601)

Western blot : Anti-SLC7A11 antibody [EPR27115-64] (ab307601) staining at 1/1000 dilution, shown in black; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 35 kDa in wild-type HepG2 cell lysates with a reduction in signal observed at this size in SLC7A11 heterozygous knockout cell line. To generate this image, wild-type and SLC7A11 heterozygous knockout HepG2 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with a high-sensitivity ECL substrate kit and imaged with 16 minutes exposure time. Secondary antibodies used were Goat anti-Rabbit HRP (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/1000 dilution

Lane 1:

Wild-type HepG2 Vehicle Control Diethyl Maleate (0mM, 24h) cell lysate at 20 µg

Lane 2:

Wild-type HepG2 Treated Diethyl Maleate (0.1mM, 24h) cell lysate at 20 µg

Lane 3:

SLC7A11 heterozygous knockout HepG2 Vehicle Control Diethyl Maleate (0mM, 24h) cell lysate at 20 µg

Lane 4:

SLC7A11 heterozygous knockout HepG2 Treated Diethyl Maleate (0.1mM, 24h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit HRP (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 35 kDa

false

• WB

Supplier Data

Western blot - Anti-xCT antibody [EPR27115-64] (AB307601)

Blocking buffer and concentration :  5% NFDM/TBST.  Diluting buffer and concentration :  5% NFDM/TBST.

Boiling leads to the formation of aggregates of membrane proteins, leading to unsuccessful migration in gel. Please do not boil your lysate when testing membrane protein.

No xCT expression or very low levels were found in lung, heart, liver, and kidney. (PMID : 22667998).

The molecular weight observed is consistent with what has been described in the literature (PMID : 26494316, 18806690, 25384799, 30425240, 29693305).

ab181602 was used as a loading control.

All lanes:

Western blot - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/1000 dilution

Lanes 1 and 6:

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

Lanes 2 and 7:

Mouse cerebellum tissue lysate at 20 µg

Lanes 3 and 8:

Mouse colon tissue lysate at 20 µg

Lanes 4 and 9:

Mouse kidney tissue lysate at 20 µg

Lanes 5 and 10:

Mouse heart tissue 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: 55 kDa

Observed band size: 37 kDa

false

Exposure time: 140s

• WB

Supplier Data

Western blot - Anti-xCT antibody [EPR27115-64] (AB307601)

Blocking and dilution buffer : 5% NFDM/TBST.

xCT protein is easy to form aggregates generating strong signal at the top.

All lanes:

Western blot - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/1000 dilution

Lanes 1 and 4:

293T cells transfected with an empty vector containing a His tag whole cell lysate at 20 µg

Lanes 2 and 5:

293T cells transfected with a Human SLC7A5 expression vector containing a His-tag whole cell lysate at 20 µg

Lanes 3 and 6:

293T cells transfected with a Human xCT expression vector containing a His-tag whole cell 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: 55 kDa

Observed band size: 37 kDa

false

Exposure time: 7s

• WB

Supplier Data

Western blot - Anti-xCT antibody [EPR27115-64] (AB307601)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Low expression cell line : COLO 205 (PMID : 31000598). The molecular weight observed is consistent with what has been described in the literature (PMID : 26494316, 18806690, 25384799, 30425240, 29693305).

All lanes:

Western blot - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

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

Lane 3:

COLO 205 (human colon adenocarcinoma tumor ascitic epithelial cell) whole cell lysate at 20 µg

Lane 4:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell 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

Observed band size: 37 kDa

false

Exposure time: 70s

• WB

Lab

Western blot - Anti-xCT antibody [EPR27115-64] (AB307601)

Western blot : Anti-SLC7A11 antibody [EPR27115-64] (ab307601) staining at 1/1000 dilution, shown in black; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab307601 was shown to bind specifically to SLC7A11. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with a high-sensitivity ECL substrate kit and imaged with 30 seconds exposure time. Secondary antibodies used were Goat anti-Rabbit HRP (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/1000 dilution

All lanes:

Recombinant Human xCT protein cell lysate at 0.01 µg

Secondary

All lanes:

Goat anti-Rabbit HRP (H+L) and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 35 kDa

false

• WB

Supplier Data

Western blot - Anti-xCT antibody [EPR27115-64] (AB307601)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : skeletal muscle (PMID : 17401668).

The molecular weight observed is consistent with what has been described in the literature (PMID : 26494316, 18806690, 25384799, 30425240, 29693305).

All lysates were not boiled. For getting better result, we recommend not boiling the sample

All lanes:

Western blot - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/1000 dilution

Lane 1:

Mouse cerebellum tissue lysate at 20 µg

Lane 2:

Mouse skeletal muscle tissue lysate at 20 µg

Lane 3:

Rat cerebellum tissue lysate at 20 µg

Lane 4:

Rat skeletal muscle tissue 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

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-xCT antibody [EPR27115-64] (AB307601)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lysates were not boiled. For getting better result we recommend not boiling the sample. The tissue samples were kindly provided by GemPharmatech. C57BL/6JGpt wildtype mice and Slc7a11-KO homozygous mice (Strain ID : T015610).

All lanes:

Western blot - Anti-xCT antibody [EPR27115-64] (ab307601) at 1/1000 dilution

Lane 1:

Wild-type mouse brain tissue lysate (male) at 40 µg

Lane 2:

Slc7a11 knockout mouse brain tissue lysate (male case1) at 40 µg

Lane 3:

Slc7a11 knockout mouse brain tissue lysate (male case2) at 40 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

Observed band size: 35 kDa

false

Exposure time: 26s