Anti-Nectin-4 antibody [RM1257]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nectin-4 antibody [RM1257] (AB322927)

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling Nectin-4 with ab322927 at 1/2000 (0.265 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human cardiac muscle.
The section was incubated with ab322927 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-Nectin-4 antibody [RM1257] (AB322927)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling Nectin-4 with ab322927 at 1/2000 (0.265 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control : no staining on human spleen.
The section was incubated with ab322927 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-Nectin-4 antibody [RM1257] (AB322927)

Flow cytometric analysis of T-47D (human ductal breast epithelial tumor epithelial cell) (Right) / A549 (human lung carcinoma epithelial cell) (Left) cells labelling Nectin-4 with ab322927 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Low expression : A549 (PMID : 21901103).

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-Nectin-4 antibody [RM1257] (AB322927)

Flow cytometric analysis of DLD-1 (human colorectal adenocarcinoma epithelial cell) (Right) / HeLa (human cervical adenocarcinoma epithelial cell) (Left) cells labelling Nectin-4 with ab322927 at 1/50 dilution (1ug) / Right compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cells.
Low expression : HeLa (PMID : 21901103).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nectin-4 antibody [RM1257] (AB322927)

Immunohistochemical analysis of paraffin-embedded Human skin tissue labeling Nectin-4 with ab322927 at 1/2000 (0.265 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human skin.
The section was incubated with ab322927 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-Nectin-4 antibody [RM1257] (AB322927)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling Nectin-4 with ab322927 at 1/2000 (0.265 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human placenta.
The section was incubated with ab322927 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

Supplier Data

Immunoprecipitation - Anti-Nectin-4 antibody [RM1257] (AB322927)

Nectin-4 was immunoprecipitated from 0.35 mg DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate with ab322927 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322927 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ab322927 IP in DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322927 in DLD-1 whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-Nectin-4 antibody [RM1257] (ab322927) at 1/30 dilution

All lanes:

DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate

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

false

Exposure time: 50s

• IP

Supplier Data

Immunoprecipitation - Anti-Nectin-4 antibody [RM1257] (AB322927)

Nectin-4 was immunoprecipitated from 0.35 mg T-47D whole cell lysate with ab322927 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322927 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate
Lane 2 : ab322927 IP in T-47D whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322927 in T-47D whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-Nectin-4 antibody [RM1257] (ab322927) at 1/30 dilution

All lanes:

T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate

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

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Nectin-4 antibody [RM1257] (AB322927)

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

Low expression : Spleen (PMID : 11544254).

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

All lanes:

Western blot - Anti-Nectin-4 antibody [RM1257] (ab322927) at 1/1000 dilution

Lane 1:

Human placenta tissue lysate at 20 µg

Lane 2:

Human spleen tissue lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 40-70 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-Nectin-4 antibody [RM1257] (AB322927)

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

Low expression : HeLa and A549 (PMID : 21901103).

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 15784625).

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

All lanes:

Western blot - Anti-Nectin-4 antibody [RM1257] (ab322927) at 1/1000 dilution

Lane 1:

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

Lane 2:

DLD-1 (human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate at 20 µg

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

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

Lane 7:

A431 (human epidermoid 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 1/100000 dilution

Observed band size: 40-70 kDa,36 kDa

true

Exposure time: 147s