Anti-NKCC1 抗体 [EPR26256-60]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling NKCC1 with ab303518 at 1/100 (4.89 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human colon(PMID : 17478539).The section was incubated with ab303518 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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized T84 (human colon epithelial cell) cells labelling NKCC1 with ab303518 at 1/100 (4.89 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing mainly membranous staining in T84 cell line. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/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 ug/ml dilution.

• Flow Cyt

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Flow Cytometry - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized T84 (human colon epithelial cell) cells labelling NKCC1 with ab303518 at 1/50 dilution (1ug) (Red) (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling NKCC1 with ab303518 at 1/100 (4.89 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Negative control : No staining on human liver.The section was incubated with ab303518 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

• IP

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Immunoprecipitation - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

NKCC1 was immunoprecipitated from 0.35 mg human cerebellum tissue lysate 10 ug with ab303518 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab303518. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution. Lane 1 : human cerebellum tissue lysate 10 µg (Inset) Lane 2 : ab303518 IP in human cerebellum tissue lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab303518 in human cerebellum tissue lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 minutes

All lanes:

Immunoprecipitation - Anti-NKCC1 antibody [EPR26256-60] (ab303518) at 1/30 dilution

Lane 1:

human cerebellum tissue lysate at 10 µg

Lane 2:

ab303518 IP in human cerebellum tissue lysate

Secondary

All lanes:

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

Predicted band size: 131 kDa

Observed band size: 150 kDa

false

Exposure time: 3min

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling NKCC1 with ab303518 at 1/2000 (4.89 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on rat colon.The section was incubated with ab303518 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling NKCC1 with ab303518 at 1/2000 (4.89 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on mouse colon.The section was incubated with ab303518 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

• Flow Cyt

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Flow Cytometry - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling NKCC1 with ab303518 at 1/50 dilution (1ug) (Red) (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling NKCC1 with ab303518 at 1/100 (4.89 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing membranous and cytoplasmic staining in NIH/3T3 cell line. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/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 ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

NKCC1 was immunoprecipitated from 0.35 mg rat brain tissue lysate 10 ug with ab303518 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab303518. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution. Lane 1 : rat brain tissue lysate 10 µg (Inset) Lane 2 : ab303518 IP in rat brain tissue lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab303518 in rat brain tissue lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 23 seconds.

All lanes:

Immunoprecipitation - Anti-NKCC1 antibody [EPR26256-60] (ab303518) at 1/30 dilution

Lane 1:

Rat brain tissue lysate at 10 µg

Lane 2:

ab303518 IP in rat brain tissue lysate

Secondary

All lanes:

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

Predicted band size: 131 kDa

Observed band size: 150 kDa

false

Exposure time: 23s

• IP

Supplier Data

Immunoprecipitation - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

NKCC1 was immunoprecipitated from 0.35 mg mouse brain tissue lysate 10 ug with ab303518 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab303518 at dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : mouse brain tissue lysate 10 µg (Inset) Lane 2 : ab303518 IP in mouse brain tissue lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab303518 in mouse brain tissue lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 15 seconds.

All lanes:

Immunoprecipitation - Anti-NKCC1 antibody [EPR26256-60] (ab303518) at 1/30 dilution

Lane 1:

Mouse brain tissue lysate at 10 µg

Lane 2:

ab303518 IP in mouse brain tissue lysate

Secondary

All lanes:

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

Predicted band size: 131 kDa

Observed band size: 150 kDa

false

Exposure time: 15s

• WB

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Western blot - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Blocking / diluting buffer and concentration : 5% NFDM/TBST Low expression : liver (human protein atlas database)

All lanes:

Western blot - Anti-NKCC1 antibody [EPR26256-60] (ab303518) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Human liver tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse colon cancer tissue lysate at 20 µg

Lane 5:

Mouse liver tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 131 kDa

Observed band size: 150 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

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

All lanes:

Western blot - Anti-NKCC1 antibody [EPR26256-60] (ab303518) at 1/1000 dilution

All lanes:

Rat brain tissue 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 1/50000 dilution

Predicted band size: 131 kDa

Observed band size: 150 kDa

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-NKCC1 antibody [EPR26256-60] (AB303518)

Blocking / diluting buffer and concentration : 5% NFDM/TBST This antibody does not cross-react with mouse NKCC2.

All lanes:

Western blot - Anti-NKCC1 antibody [EPR26256-60] (ab303518) at 1/1000 dilution

Lane 1:

His-tagged mouse NKCC2 recombinant protein at 10 ng

Lane 2:

His-tagged mouse NKCC1 recombinant protein at 10 ng

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 1/50000 dilution

Predicted band size: 131 kDa

Observed band size: 30 kDa

false

Exposure time: 26s