Anti-TDP43 antibody [EPR18554] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

This data was developed using ab190963, the same antibody clone in a different buffer formulation.

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190963).

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] - BSA and Azide free (AB238443)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190963).

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190963).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190963).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cell line from bone marrow) cells labeling TDP43 with 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190963).

• IP

Supplier Data

Immunoprecipitation - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190963).

All lanes:

Immunoprecipitation - Anti-TDP43 antibody [EPR18554] (<a href='/en-us/products/primary-antibodies/tdp43-antibody-epr18554-ab190963'>ab190963</a>)

Predicted band size: 44 kDa

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• WB

Supplier Data

Western blot - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab190963).

All lanes:

Western blot - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (ab238443)

Predicted band size: 44 kDa

false

• WB

Lab

Western blot - Anti-TDP43 antibody [EPR18554] - BSA and Azide free (AB238443)

This data was developed using ab190963, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/tdp43-antibody-epr18554-ab190963'>ab190963</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 50 µg

Lane 2:

TARDBP knock-out HAP1 lysate at 50 µg

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