Anti-ATP13A2 antibody [MJF-D29756-28]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Wildtype HEK-293 (human embryonic kidney epithelial cell) and ATP13A2 KO HEK-293 (ATP13A2 knockout human embryonic kidney epithelial cell) cells labelling ATP13A2 with ab322735 at 1/500 (1 µg/ml) dilution (Red).

The data was kindly provided by a collaborator.

The nuclear counterstain was DAPI (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling ATP13A2 with ab322735 at 1/100 (5.24 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human cerebrum. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

Immunohistochemical analysis of paraffin-embedded Human Parkinson's disease brain tissue labeling ATP13A2 with ab322735 at 1/100 (5.24 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on human Parkinson's disease brain. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

Immunohistochemical analysis of paraffin-embedded (A) Wildtype HEK-293 (human embryonic kidney epithelial cell) cell pellet and (B) ATP13A2 knockout HEK-293 cell pellet. tissue labeling ATP13A2 with ab322735 at 1/100 (5.24 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Positive staining on (A) Wildtype HEK-293 cell pellet, no staining on (B) ATP13A2 knockout HEK-293 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

Lab

Immunoprecipitation - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

ATP13A2 was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell) whole cell lysate with ab322735 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322735 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : ab322735 at 1/30 IP in HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322735 in HEK-293 whole cell lysate

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

Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-ATP13A2 antibody [MJF-D29756-28] (ab322735) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab322735 at 1/30 IP in HEK-293 (human embryonic kidney epithelial cell) 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 ab322735 in HEK-293 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

Observed band size: 150 kDa

false

Exposure time: 180s

• IP

Lab

Immunoprecipitation - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

ATP13A2 was immunoprecipitated from 0.35 mg Saos-2 (human osteosarcoma epithelial cell) whole cell lysate with ab322735 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322735 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Saos-2 (human osteosarcoma epithelial cell) whole cell lysate
Lane 2 : ab322735 at 1/30 IP in Saos-2 (human osteosarcoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322735 in Saos-2 whole cell lysate

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

Exposure time : 102 seconds.

All lanes:

Immunoprecipitation - Anti-ATP13A2 antibody [MJF-D29756-28] (ab322735) at 1/1000 dilution

Lane 1:

Saos-2 (human osteosarcoma epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab322735 at 1/30 IP in Saos-2 (human osteosarcoma epithelial cell) 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 ab322735 in Saos-2 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

Observed band size: 150 kDa

false

Exposure time: 102s

• WB

Lab

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

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

ab322735 was shown to react with ATP13A2 in wild-type HEK-293 cells in Western blot with loss of signal observed in ATP13A2 knockout cell line ab274925. Wild-type HEK-293 and ATP13A2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab322735 overnight at 4 °C at a 1/1000 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-ATP13A2 antibody [MJF-D29756-28] (ab322735) at 1/1000 dilution

Lane 1:

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

Lane 2:

Western blot - Human ATP13A2 knockout HEK-293 cell lysate (<a href='/en-us/products/cell-lysates/human-atp13a2-knockout-hek-293-cell-lysate-ab274983'>ab274983</a>) 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

Observed band size: 150 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

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

In Western blot, ab322735 was shown to bind specifically to ATP13A2. Target of interest was observed at 150 kDa in wild-type HEK-293 cell lysates (lane 1) with no signal observed at this size in ATP13A2 knockout cell line (lane 2)(knockout cell line ab274925)(knockout lysate ab274983)

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1 : 100000 (15Kda).

All lanes:

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (ab322735) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 50 µg

Lane 2:

Western blot - Human ATP13A2 knockout HEK-293 cell lysate (<a href='/en-us/products/cell-lysates/human-atp13a2-knockout-hek-293-cell-lysate-ab274983'>ab274983</a>) at 50 µg

Secondary

All lanes:

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

Observed band size: 150 kDa,15 kDa

false

Exposure time: 92s

• WB

Lab

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

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

Low expression : SK-N-BE(2), lung, skeletal muscle

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 26 seconds, lane 3 : 92 seconds, lanes 4-5 : 103 seconds

All lanes:

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (ab322735) at 1/1000 dilution

Lane 1:

Saos-2 (human osteosarcoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

SK-N-BE(2) (human neuroblastoma neuroblast) whole cell lysate at 50 µg

Lane 3:

Human cerebellum tissue lysate at 50 µg

Lane 4:

Human hypothalamus tissue lysate at 50 µg

Lane 5:

Human skeletal muscle tissue lysate at 50 µg

Lane 6:

Human lung muscle tissue lysate at 50 µg

Secondary

All lanes:

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

Observed band size: 150 kDa,36 kDa

false

• WB

Collaborator

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

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

The data was kindly provided by a collaborator.

All lanes:

Western blot - Anti-ATP13A2 antibody [MJF-D29756-28] (ab322735) at 1/1000 dilution

Lane 1:

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

Lane 2:

Hela transfected with siRNA specifically targeting ATP13A2 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

Observed band size: 150 kDa

false

Exposure time: 180s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

Immunohistochemical analysis of paraffin-embedded Human lung tissue labeling ATP13A2 with ab322735 at 1/100 (5.24 µg/ml) dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Negative control : no staining on human lung. 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATP13A2 antibody [MJF-D29756-28] (AB322735)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293T (human embryonic kidney epithelial cell) transfected with a human ATP13A2 expression vector containing a Myc-His tag and (B) HEK-293T transfected with empty vector containing a Myc-His tag tissue labeling ATP13A2 with ab322735 at 1/2000 (0.262 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining on HEK-293T transfected with a Myc-His-tagged human ATP13A2 construct, no staining on (B) HEK-293T transfected with empty vector containing a Myc-His tag. 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) was used.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins