Anti-SLC7A5/LAT1 antibody [EPR26260-66]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (AB305251)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized LNCaP (human prostate carcinoma epithelial cell, Left) / HT-29 (human colorectal adenocarcinoma epithelial cell, Right) cells labelling SLC7A5/LAT1 with ab305251 at 1/500 dilution (0.1ug) (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. Negative control : LNCaP (PMID : 30241549).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (AB305251)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HT-29 (human colorectal adenocarcinoma epithelial cell) cells labelling SLC7A5/LAT1 with ab305251 at 1/100 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in HT-29 cell line. Negative control : LNCaP (PMID : 30241549). 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 (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 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (AB305251)

Immunohistochemical analysis of paraffin-embedded Human lung squamous tissue labeling SLC7A5/LAT1 with ab305251 at 1/500 (1.03 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Membranous staining on human lung squamous carcinoma. The section was incubated with ab305251 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval 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-SLC7A5/LAT1 antibody [EPR26260-66] (AB305251)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling SLC7A5/LAT1 with ab305251 at 1/500 (1.03 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Membranous staining on human testis (PMID : 35050188). The section was incubated with ab305251 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (AB305251)

SLC7A5/LAT1 was immunoprecipitated from 0.35 mg HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate 10 μg with ab305251 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305251. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate 10 μg Lane 2 : ab305251 IP in HT-29 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab305251 in HT-29 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 seconds Samples are non-boiled as boiling may cause protein aggregates.

All lanes:

Immunoprecipitation - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (ab305251) at 1/30 dilution

All lanes:

HT-29 (human colorectal adenocarcinoma 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: 40 kDa

false

Exposure time: 3s

• WB

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Western blot - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (AB305251)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : LNCaP (PMID : 30241549) Samples are non-boiled as boiling may cause protein aggregates (multi-pass membrane protein) Exposure time : 136 seconds

All lanes:

Western blot - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (ab305251) at 1/1000 dilution

Lane 1:

HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

LNCaP (human prostate carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma 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 1/100000 dilution

Observed band size: 40 kDa

false

Exposure time: 136s

• WB

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Western blot - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (AB305251)

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

Exposure time : 1 second, left; 3 seconds, right.

All lanes:

Western blot - Anti-SLC7A5/LAT1 antibody [EPR26260-66] (ab305251) at 1/1000 dilution

Lanes 1 and 3:

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

Lanes 2 and 4:

293T cells transfected with a Human SLC7A5 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: 1s