Anti-ADNP antibody [EPR25434-4] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labelling ADNP with ab300114 at 1/100 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human colon carcinoma is observed (PMID : 27903678). The section was incubated with ab300114 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed and 90% methnol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling ADNP with ab300114 at 1/500 dilution (Red) compared with a rabbit monoclonal IgG (ab172730) (Black) isotype control. Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

This data was developed using ab300114, the same antibody clone in a different buffer formulation.

ab300114 was shown to react with ADNP in wild-type DMS 53 cells in immunocytochemistry with loss of signal observed in a ADNP siRNA knockdown cell line. Wild-type and siRNA knockdown cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab300114 at 1/500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (siRNA knockdown) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and siRNA knockdown cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• ICC

Supplier Data

Immunocytochemistry - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeilized HeLa (human cervix adenocarcinoma epithelial cell) cells lebelling ADNP with ab300114 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in HeLa cell line. Tubuline was stained with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : PBS was used instead of ab300114 followed by preabsorbed secondary antibody (ab150081) Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

ADNP was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab300114 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab300114 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution. Lane 1 (Input) : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg. Lane 2 (+) : ab300114 IP in HeLa whole cell lysate. Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab300114 in HeLa whole cell lysate. Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 84 seconds

All lanes:

Immunoprecipitation - Anti-ADNP antibody [EPR25434-4] (<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a>) at 1/30 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a> IP in HeLa whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a> in HeLa whole cell lysate

false

• IP

Supplier Data

Immunoprecipitation - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

This data was developed using ab300114, the same antibody clone in a different buffer formulation.

Immunoprecipitation of ADNP in DMS 53 cells. Lysates were prepared and immunoprecipitation was performed using 2 µg of ab300114 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-ADNP antibody [EPR25434-4] (<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a>) at 2 µg

Lanes 1 and 3:

DMS 53 cells

Lane 2:

Unbound fraction: Rabbit monoclonal IgG instead of <a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a> in DMS 53 cells

false

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum (fresh) tissue labeling ADNP with ab300114 at 1/500 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (Green). Positive staining on rat cerebrum is observed. The nuclear counterstain was DAPI (Blue). PBS was used instead of ab300114 followed by preabsorbed secondary antibody (ab150081) Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• ICC

Supplier Data

Immunocytochemistry - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

This data was developed using ab300114, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeilized NIH/3T3 (mouse embryonic fibroblast) cells lebelling ADNP with ab300114 at 1/50 (11.22 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2µg/mL) dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody control : PBS was used instead of ab300114 followed by preabsorbed secondary antibody (ab150081) Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 (2 µg/mL) dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Immunohistochemical analysis of paraffin-embedded mouse glioblastoma tissue labelling ADNP with ab300114 at 1/100 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse glioblastoma is observed. The section was incubated with ab300114 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval 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-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labelling ADNP with ab300114 at 1/100 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat cerebrum is observed. The section was incubated with ab300114 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling ADNP with ab300114 at 1/500 dilution (Red) compared with a rabbit monoclonal IgG (ab172730) (Black) isotype control. A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum (fresh) tissue labeling ADNP with ab300114 at 1/500 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (Green). Positive staining on mouse cerebrum is observed. The nuclear counterstain was DAPI (Blue). Secondary antibody control : PBS was used instead of ab300114 followed by preadsorbed secondary antibody (ab150081) Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling ADNP with ab300114 at 1/100 dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Nuclear staining on mouse cerebrum is observed. The section was incubated with ab300114 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin. Secondary antibody only control : PBS was used instead of primary antibody followed by ready to use secondary antibody LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

Supplier Data

Immunoprecipitation - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

ADNP was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab300114 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab300114 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution. Lane 1 (Input) : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 µg. Lane 2 (+) : ab300114 IP in NIH/3T3 whole cell lysate. Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab300114 in NIH/3T3 whole cell lysate. Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-ADNP antibody [EPR25434-4] (<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a>) at 1/30 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a> IP in NIH/3T3 whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a> in NIH/3T3 whole cell lysate

false

• WB

Supplier Data

Western blot - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

This data was developed using ab300114, the same antibody clone in a different buffer formulation.

ab300114 was shown to react with ADNP in wild-type DMS 53 cells in Western blot with loss of signal observed in a ADNP siRNA knockdown cell line. Cell lysates from wild-type DMS 53 transfected with either scrambled siRNA or ADNP siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab300114 overnight at 4 °C at a 1/10000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-ADNP antibody [EPR25434-4] (<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a>) at 1/10000 dilution

Lane 1:

Wild-type DMS 53 transfected with scrambled siRNA control lysate at 30 µg

Lane 2:

DMS 53 transfected with siRNA specifically targeting ADNP cell lysate at 30 µg

false

• WB

Supplier Data

Western blot - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Blocking and dilution buffer and concentration : 5% NFDM/TBST. Lysates should be made freshly and used in WB immediately to minimize protein degradation.

All lanes:

Western blot - Anti-ADNP antibody [EPR25434-4] (<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a>) at 1/1000 dilution

Lane 1:

C6 (Rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 2:

U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Neuro-2a (Mouse neuroblastoma neuroblast) 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

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

This data was developed using ab300114, the same antibody clone in a different buffer formulation.

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

Exposure time :

Lanes 1-3 : 114 seconds; Lane 4 : 59 seconds.

Lysates should be made freshly and used in WB immediately to minimize protein degradation.

All lanes:

Western blot - Anti-ADNP antibody [EPR25434-4] (<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix 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: 124 kDa

Observed band size: 140 kDa

false

• WB

Supplier Data

Western blot - Anti-ADNP antibody [EPR25434-4] - BSA and Azide free (AB300115)

Blocking and dilution buffer and concentration : 5% NFDM/TBST. Lysates should be made freshly and used in WB immediately to minimize protein degradation. We used fresh mouse brain tissue lysate. This blot was developed using a higher sensitivity ECL substrate.

All lanes:

Western blot - Anti-ADNP antibody [EPR25434-4] (<a href='/en-us/products/primary-antibodies/adnp-antibody-epr25434-4-ab300114'>ab300114</a>) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain 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 1/20000 dilution

false

Exposure time: 3s