Anti-TDP43 antibody [EPR18554]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR18554] (AB190963)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on K562 cell line.

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab190963 at 1/2000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-TDP43 antibody [EPR18554] (AB190963)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling TDP43with ab190963 at 1/1000 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] -Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR18554] (AB190963)

Immunohistochemical analysis of paraffin-embedded Human endometrium carcinoma tissue labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on tumor cells of the endometrium carcinoma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR18554] (AB190963)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on normal Human pancreas is observed.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR18554] (AB190963)

ab190963 was shown to react with TARDBP in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a TARDBP knockout cell line. Wild-type and knockout 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 ab190963 at  1/1000 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 (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout 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).

These data were 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/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR18554] (AB190963)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling TDP43 with ab190963 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining on HeLa cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab190963 at 1/2000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-TDP43 antibody [EPR18554] (AB190963)

TDP43 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab190963 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab190963 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate, 10μg (Input).

Lane 2 : ab190963 IP in HeLa whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab190963 in HeLa whole cell lysate.

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

Exposure time : 1 second.

The band of ~35 kDa is the cleavage product from caspase activity (PMID : 22659571, PMID : 20736350).

All lanes:

Immunoprecipitation - Anti-TDP43 antibody [EPR18554] (ab190963)

Predicted band size: 44 kDa

false

• WB

Lab

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

ab190963 was shown to react with TARDBP in wild-type HAP1 cells in Western blot with loss of signal observed in a TARDBP knockout cell line. Wild-type HAP1 and TARDBP knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab190963 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were 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-TDP43 antibody [EPR18554] (ab190963) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 50 µg

Lane 2:

TARDBP knock-out HAP1 lysate at 50 µg

false

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking/Dilution buffer : 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID : 22659571, PMID : 20736350).

All lanes:

Western blot - Anti-TDP43 antibody [EPR18554] (ab190963) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Secondary

All lanes:

Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 44 kDa

Observed band size: 45 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking/Dilution buffer : 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID : 22659571, PMID : 20736350).

All lanes:

Western blot - Anti-TDP43 antibody [EPR18554] (ab190963) at 1/10000 dilution

Lane 1:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate lysate at 20 µg

Lane 2:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate 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

Predicted band size: 44 kDa

Observed band size: 45 kDa

false

Exposure time: 5s

• WB

Lab

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : TDP43 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab190963 observed at 48 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab190963 was shown to specifically react with TDP43 in wild type cells as signal was lost in TDP43 knockout cells. Wild-type and TDP43 knockout samples were subjected to SDS-PAGE. ab190963 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-TDP43 antibody [EPR18554] (ab190963)

Predicted band size: 44 kDa

false

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking/Dilution buffer : 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID : 22659571, PMID : 20736350), and can be induced by staurosporine.

All lanes:

Western blot - Anti-TDP43 antibody [EPR18554] (ab190963) at 1/1000 dilution

Lane 1:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 1μM staurosporine for 4 hours whole cell lysate at 20 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 50 μM Z-VAD-FMK for 4 hours whole cell lysate at 20 µg

Lane 4:

HeLa (Human epithelial cell line from cervix adenocarcinoma) treated with 1μM staurosporine and 50 μM Z-VAD-FMK for 4 hours 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

Predicted band size: 44 kDa

Observed band size: 45 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-TDP43 antibody [EPR18554] (ab190963) at 1/2000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) 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

Predicted band size: 44 kDa

Observed band size: 45 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-TDP43 antibody [EPR18554-55] (ab190963) staining at 1/1000 dilution In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution

All lanes:

Western blot - Anti-TDP43 (phospho S409 + pS410) antibody [EPR24512-120] (<a href='/en-us/products/primary-antibodies/tdp43-phospho-s409-ps410-antibody-epr24512-120-ab314299'>ab314299</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T cells transfected with a human TDP43 expression vector containing a His-tag, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

293T cells transfected with a human TDP43 expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate (untreated membrane) at 20 µg

Lane 4:

293T cells transfected with a human TDP43 expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate (Alkaline phosphatase treated membrane) 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: 50 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The antibody specifically recognizes TDP43 diphosphorylated at S409 and S410. It does not cross-react with TDP43 singly phosphorylated at S409 or singly phosphorylated at S410. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-TDP43 antibody [EPR18554-55] (ab190963) staining at 1/1000 dilution In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution

All lanes:

Western blot - Anti-TDP43 (phospho S409 + pS410) antibody [EPR24512-120] (<a href='/en-us/products/primary-antibodies/tdp43-phospho-s409-ps410-antibody-epr24512-120-ab314299'>ab314299</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T cells transfected with a human wild-type TDP43 expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human wild-type TDP43 expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human TDP43 (S409A and S410A mutation) expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a human TDP43 (S409A mutation) expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate at 20 µg

Lane 6:

293T cells transfected with a human TDP43 (S410A mutation) expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, 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: 50 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The antibody specifically recognizes TDP43 singly phosphorylated at S410. In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-TDP43 antibody [EPR18554-55] (ab190963) staining at 1/1000 dilution. In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-TDP43 (phospho S410) antibody [EPR24512-8] (<a href='/en-us/products/primary-antibodies/tdp43-phospho-s410-antibody-epr24512-8-ab314301'>ab314301</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T cells transfected with a human wild-type TDP43 expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human wild-type TDP43 expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human TDP43 (S409A and S410A mutation) expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a human TDP43 (S409A mutation) expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate at 20 µg

Lane 6:

293T cells transfected with a human TDP43 (S410A mutation) expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, 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: 50 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-TDP43 antibody [EPR18554-55] (ab190963) staining at 1/1000 dilution. In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-TDP43 (phospho S410) antibody [EPR24512-8] (<a href='/en-us/products/primary-antibodies/tdp43-phospho-s410-antibody-epr24512-8-ab314301'>ab314301</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T cells transfected with a human TDP43 expression vector containing a His-tag, whole cell lysate (untreated membrane) at 20 µg

Lane 3:

293T cells transfected with a human TDP43 expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate (untreated membrane) at 20 µg

Lane 4:

293T cells transfected with a human TDP43 expression vector containing a His-tag were treated with 1 uM Okadaic Acid for 60 minutes, whole cell lysate (Alkaline phosphatase treated membrane) 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: 50 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] (AB190963)

Blocking/Dilution buffer : 5% NFDM/TBST.

The band of ~35 kDa is the cleavage product from caspase activity (PMID : 22659571, PMID : 20736350).

All lanes:

Western blot - Anti-TDP43 antibody [EPR18554] (ab190963) at 1/1000 dilution

All lanes:

Zebrafish head lysate at 10 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

Predicted band size: 44 kDa

Observed band size: 45 kDa

false

Exposure time: 3min