Recombinant 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, PMID: 18806690).

ab181602 was used as a loading control.

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, PMID: 18806690).

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, PMID: 18806690).

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling xCT with ab307601 at 1/500 (1.024 µg/ml) followed by a 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 a 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.

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

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling xCT with ab307601 at 1/500 (1.024 µg/ml) followed by a 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 a 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.

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling xCT with ab307601 at 1/500 (1.024 µg/ml) followed by a 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 a 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.

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling xCT with ab307601 at 1/500 (1.024 µg/ml) followed by a 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 a 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.

Immunohistochemical analysis of paraffin-embedded Mouse skeletal muscle tissue labeling xCT with ab307601 at 1/500 (1.024 µg/ml) followed by a 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 a 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.

Immunohistochemical analysis of paraffin-embedded Rat skeletal muscle tissue labeling xCT with ab307601 at 1/500 (1.024 µg/ml) followed by a 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 a 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.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labelling xCT with ab307601 at 1/500 (1.024 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 µg/ml) 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 (2.5 µg/ml) dilution (Red). The 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 (2 µg/ml) dilution.

xCT was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) whole cell lysate 10 µg with ab307601 at 1/30 dilution (2µg in 0.35mg 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 (human lung carcinoma epithelial cell) 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

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized COLO 205 (human colon adenocarcinoma tumor ascitic epithelial cell, Left) / A549 (human lung carcinoma epithelial cell, Right) cells labelling xCT with ab307601 at 1/50 dilution (1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled 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).