Anti-NEDD4 antibody [EPR30127-570]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded (A) Normal human lung. (B) Human lung carcinoma. tissue labeling NEDD4 with ab324563 at 1/200 (2.57 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining observed in (A) normal human lung, with increased expression of NEDD4 in (B) lung carcinoma tissue (PMID : 20889565).
The primary antibody was incubated 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-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded Human mammary gland tissue labeling NEDD4 with ab324563 at 1/200 (2.57 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human mammary gland (PMID : 31856858).
The primary antibody was incubated 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-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) cell pellet. (B) NEDD4 knockout HAP1 cell pellet. tissue labeling NEDD4 with ab324563 at 1/200 (2.57 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) Wild-type HAP1 cell pellet, no staining on (B) NEDD4 knockout HAP1 cell pellet.
The primary antibody was incubated 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-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded Human thyroid carcinoma tissue labeling NEDD4 with ab324563 at 1/200 (2.57 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human thyroid carcinoma.
The primary antibody was incubated 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-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling NEDD4 with ab324563 at 1/200 (2.57 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cervical carcinoma.
The primary antibody was incubated 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-Fr

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Immunohistochemistry (Frozen sections) - Anti-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse cerebrum (perfused fixed) tissue labeling NEDD4 with ab324563 at 1/50 (10.28 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining on mouse cerebrum.
Panel A : merged staining of anti-NEDD4 (ab324563, green), anti-NeuN (ab190565, magenta) on mouse cerebrum.
Panel B : anti-NEDD4 stained on mouse cerebrum.
Panel C : anti-NeuN stained in neurons of mouse cerebrum.
The section was incubated in two rounds of staining : in the order of ab324563 and ab190565, for 1 hr at room temperature. The nuclear counterstain was DAPI (Blue). The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 2 ug/mL dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20)

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded Mouse lung adenocarcinoma tissue labeling NEDD4 with ab324563 at 1/4000 (0.129 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse lung adenocarcinoma.
The primary antibody was incubated for 10 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling NEDD4 with ab324563 at 1/4000 (0.129 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse liver (PMID : 9073511).
The primary antibody was incubated for 10 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-NEDD4 antibody [EPR30127-570] (AB324563)

Immunohistochemical analysis of paraffin-embedded Mouse breast carcinoma tissue labeling NEDD4 with ab324563 at 1/4000 (0.129 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse breast carcinoma.
The primary antibody was incubated for 10 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Slides were incubated with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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

• WB

Supplier Data

Western blot - Anti-NEDD4 antibody [EPR30127-570] (AB324563)

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

The samples are unboiled.

The bands beneath the target band (115 kDa) are likey to be degradation products.

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

All lanes:

Western blot - Anti-NEDD4 antibody [EPR30127-570] (ab324563) at 1/1000 dilution

Lane 1:

Human breast tissue lysate at 40 µg

Lane 2:

Human tonsil tissue lysate at 40 µg

Secondary

All lanes:

Goat anti-Mouse IgG Fc Cross-Adsorbed Secondary Antibody, HRP (ThermoFisher, 31439) at 1/2000 dilution

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-NEDD4 antibody [EPR30127-570] (AB324563)

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

Performed under reducing conditions.

In Western blot, ab324563 was shown to bind specifically to NEDD4. Target of interest was observed at 115 kDa in wild-type HAP1 cell lysates (lane 2) with no signal observed at this size in NEDD4 knockout cell line (lane 1) (lane 1, knockout cell line ab

ab256783).

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The samples are unboiled.

All lanes:

Western blot - Anti-NEDD4 antibody [EPR30127-570] (ab324563) at 1/1000 dilution

Lane 1:

NEDD4 knockout parental HAP1 (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate at 20 µg

Lane 2:

Parental HAP1 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

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-NEDD4 antibody [EPR30127-570] (AB324563)

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

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

The samples are unboiled.

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

All lanes:

Western blot - Anti-NEDD4 antibody [EPR30127-570] (ab324563) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

NIH/3T3 transfected with siRNA specifically targeting NEDD4 whole cell lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

Mouse testis tissue lysate at 20 µg

Lane 6:

Mouse spleen tissue 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

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-NEDD4 antibody [EPR30127-570] (AB324563)

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

This antibody does not cross-react with NEDD4L 4 by western blot.

All lanes:

Western blot - Anti-NEDD4 antibody [EPR30127-570] (ab324563) at 1/1000 dilution

Lane 1:

His-tagged human NEDD4 fragment at 10 ng

Lane 2:

No-tagged human NEDD4L fragment at 10 ng

Secondary

All lanes:

Goat anti-Mouse IgG Fc Cross-Adsorbed Secondary Antibody, HRP (ThermoFisher, 31439) at 1/2000 dilution

false

Exposure time: 26s