Anti-Nectin-4 antibody [EPR15613-68] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nectin-4 antibody [EPR15613-68] - BSA and Azide free (AB251110)

This data was developed using ab192033, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of formalin fixed paraffin (FFPE) embedded normal human skin labelling Nectin-4 with ab192033 at a concentration of 0.3μg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit followed by OptiView Amplification kit. Heat mediated antigen retrieval was conducted for 64min with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5. ab192033 was incubated at 37°C for 40min. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers should note, the current antibody has been examined with alternative automated IHC staining platforms and optimal results were obtained using the Ventana DISCOVERY ULTRA with associated kits and reagents. We were not able to obtain optimal results with Leica BOND platform when using the Polymer Refine detection kit, 20min ER1 antigen retrieval and 16min antibody incubation at room temperature. Further optimisation may be needed when using the Leica BOND platform.

Some nuclear non-specific staining can be observed in Ventana when antibody concentration >1 ug/mL.

• WB

Supplier Data

Western blot - Anti-Nectin-4 antibody [EPR15613-68] - BSA and Azide free (AB251110)

This data was developed using ab192033, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. The expression profile observed in MDA-MB-231 is consistent with the literatures (PMID : 31617074, PMID : 24386110, PMID : 17474988). Negative control : MDA-MB-231 (PMID : 31617074, PMID : 24386110, PMID : 17474988).

All lanes:

Western blot - Anti-Nectin-4 antibody [EPR15613-68] (<a href='/en-us/products/primary-antibodies/nectin-4-antibody-epr15613-68-ab192033'>ab192033</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Human placenta tissue lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 55 kDa

Observed band size: 40-70 kDa

false

Exposure time: 20s

• WB

Lab

Western blot - Anti-Nectin-4 antibody [EPR15613-68] - BSA and Azide free (AB251110)

This data was developed using ab192033, the same antibody clone in a different buffer formulation.

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

ab181602 was used as a GAPDH loading control.

The antibody detected a 50kDa and 70kDa non-specific band in the negative sample MDA-MB-231, which are close to the target band. Therefore, we recommend including a negative sample when performing Western Blot analysis.

We suggest using the whole cell and cell supernatant lysate side by side to observe the expression of different form of Nectin-4. The cell supernatant can detect soluble Nectin-4 mainly (PMID : 15784625).

Negative control : MDA-MB-231 (15784625, PMID : 31617074, PMID : 24386110, PMID : 17474988).

All lanes:

Western blot - Anti-Nectin-4 antibody [EPR15613-68] (<a href='/en-us/products/primary-antibodies/nectin-4-antibody-epr15613-68-ab192033'>ab192033</a>) at 1/1000 dilution

Lane 1:

T-47D (Human ductal breast epithelial tumor epithelial cell) starved for 24 hours, whole cell lysate at 20 µg

Lane 2:

T-47D (Human ductal breast epithelial tumor epithelial cell) starved for 24 hours, Concentrated cell supernatant at 20 µg

Lane 3:

MDA-MB-231 (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 1/20000 dilution

Predicted band size: 55 kDa

Observed band size: 40-70 kDa

false

Exposure time: 7s

• WB

Supplier Data

Western blot - Anti-Nectin-4 antibody [EPR15613-68] - BSA and Azide free (AB251110)

This data was developed using ab192033, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Nectin-4 antibody [EPR15613-68] (<a href='/en-us/products/primary-antibodies/nectin-4-antibody-epr15613-68-ab192033'>ab192033</a>) at 1/1000 dilution

Lane 1:

T47D lysate at 20 µg

Lane 2:

MCF7 lysate at 20 µg

Lane 3:

Human placenta lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 55 kDa

false