Anti-ATAD3A antibody [EPR27484-26]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling ATAD3A with ab317252 at 1/1000 (0.506 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast cancer (PMID : 36627348).
The section was incubated with ab317252 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-ATAD3A antibody [EPR27484-26] (AB317252)

Immunohistochemical analysis of paraffin-embedded Human colorectal carcinoma tissue labeling ATAD3A with ab317252 at 1/1000 (0.506 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colorectal carcinoma.
The section was incubated with ab317252 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-ATAD3A antibody [EPR27484-26] (AB317252)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling ATAD3A with ab317252 at 1/50 (10.12 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 mitochondrial staining in HeLa cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain at 1/1000 (1ug/ml) dilution (Magenta). 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 (Intra)

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Flow Cytometry (Intracellular) - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling ATAD3A with ab317252 at 1/500 dilution (0.1 ug)/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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling ATAD3A with ab317252 at 1/1000 (0.506 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cerebrum (PMID : 35236834).
The section was incubated with ab317252 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-ATAD3A antibody [EPR27484-26] (AB317252)

ATAD3A was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab317252 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317252 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ab317252 IP in HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317252 in HeLa whole cell lysate

All lanes:

Immunoprecipitation - Anti-ATAD3A antibody [EPR27484-26] (ab317252) at 1/30 dilution

All lanes:

HeLa (human cervix 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: 6s

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling ATAD3A with ab317252 at 1/500 dilution (0.1 ug)/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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling ATAD3A with ab317252 at 1/50 (10.12 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 mitochondrial staining in NIH/3T3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain at 1/1000 (1ug/ml) dilution (Magenta). 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.

• WB

Supplier Data

Western blot - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

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

In Western blot, Anti-TOMM20 antibody [EPR15581-54] - Mitochondrial Marker (ab186735) staining at 1/2000 dilution.

All lanes:

Western blot - Anti-ATAD3A antibody [EPR27484-26] (ab317252) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) non-mitochondrial fraction at 20 µg

Lane 2:

HeLa mitochondria fraction 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: 66 kDa,36 kDa,16 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

Lanes 2-7 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 and lane 1 is applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

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

Exposure time : Lane 1 : 26 seconds, lanes 2-7 : 180 seconds

All lanes:

Western blot - Anti-ATAD3A antibody [EPR27484-26] (ab317252) at 1/1000 dilution

Lane 1:

Human cerebellum tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse pancreas tissue lysate at 20 µg

Lane 5:

Rat heart tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

Rat spleen tissue lysate at 20 µg

Secondary

Lane 1:

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

Lanes 2 - 7:

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: 66 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

This antibody recognizes overexpressed human ATAD3A isoform 2 and isoform 1 (UniProt ID : Q9NVI7).

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

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-ATAD3A antibody [EPR27484-26] (ab317252) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a Human ATAD3A isoform 2 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a Human ATAD3A isoform 1 expression vector containing a myc-His-tag®, 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: 66 kDa,76 kDa,36 kDa

false

Exposure time: 62s

• WB

Supplier Data

Western blot - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

The lane 8 was developed using a high sensitivity ECL substrate.

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

Exposure time : Lanes 1-4 : 26 seconds, lanes 5-8 : 180 seconds

All lanes:

Western blot - Anti-ATAD3A antibody [EPR27484-26] (ab317252) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeting ATAD3A whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Lane 5:

MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate at 20 µg

Lane 6:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

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

Lane 8:

NIH/3T3 (mouse embryonic fibroblast) 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: 66 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-ATAD3A antibody [EPR27484-26] (AB317252)

This antibody does not cross-react with human ATAD3B and ATAD3C.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-ATAD3A antibody [EPR27484-26] (ab317252) at 1/1000 dilution

Lane 1:

His-tagged human ATAD3A fragment at 40 ng

Lane 2:

His-tagged human ATAD3B fragment at 40 ng

Lane 3:

His-tagged human ATAD3C fragment at 40 ng

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: 36 kDa

false

Exposure time: 180s