Anti-TDP43 antibody [EPR5810] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

This data was developed using ab109535, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human Alzheimer's brain tissue* labelling TDP43 with ab109535 at 0.1ug/ml followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Positive staining in human Alzheimer's brain.

The section was incubated with ab109535 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 with sodium citrate buffer (pH 6.0 epitope retrieval solution1) for 20 mins.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• WB

Lab

Western blot - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

This data was developed using ab109535, the same antibody clone in a different buffer formulation.

ab109535 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 ab109535 overnight at 4 °C at a 1/2000 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 [EPR5810] (<a href='/en-us/products/primary-antibodies/tdp43-antibody-epr5810-ab109535'>ab109535</a>) at 1/2000 dilution

Lane 1:

Wild-type HAP1 lysate at 50 µg

Lane 2:

TARDBP knock-out HAP1 lysate at 50 µg

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• WB

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Western blot - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

All lanes:

Western blot - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (ab185133) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

K562 cell lysate at 10 µg

Lane 4:

A431 cell lysate at 10 µg

Secondary

All lanes:

HRP-labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 44 kDa

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• WB

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Western blot - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

All lanes:

Western blot - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (ab185133) at 1/5000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

Mouse brain lysates at 15 µg

Lane 3:

Rat brain lysates at 15 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: 44 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

IHC image of TDP43 staining in a section of frozen human prostate carcinoma performed on a Leica BOND^TM system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab109535, 1/100 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling TDP43 with purified ab109535 at 1/50 dilution (6.2 μg/ml). Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with ab195889 anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109535).

This antibody is not suitable to detect TDP43 using PFA fixation in ICC.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue sections labeling TDP43 with purified ab109535 at 1/100 dilution (0.3 μg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109535).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

ab109535 staining TDP43 in wild-type HAP1 cells (top panel) and TDP43 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab109535 at 1μg/ml concentration and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109535).

This antibody is not suitable to detect TDP43 using PFA fixation in ICC.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

ab109535 at 1/100 dilution staining TARDBP in paraffin-embedded Human papillary carcinoma tissue by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109535).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

Intracellular Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling TDP43 with purified ab109535 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109535).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labeling TDP43 with purified ab109535 at 1/100 dilution (0.3 μg/ml). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109535).

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

Immunohistochemistry (Frozen sections) analysis of mouse cerebrum tissue sections labeling TDP43 with Purified unpurified ab109535 at 1/50 (0.5 μg/ml).Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. DAPI was used as a counterstain.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109535).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

Clone EPR5810 (ab185133) has been successfully conjugated by Abcam. This image was generated using Anti-TDP43 antibody [EPR5810] (Alexa Fluor® 488). Please refer to ab193842 for protocol details.

ab193842 staining TDP43 in Hek293 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab193842 at a 1/250 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This antibody is not suitable to detect TDP43 using PFA fixation in ICC.

• WB

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Western blot - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (AB185133)

Lane 1 : Wild-type HAP1 cell lysate (40 μg)
Lane 2 : TDP43 knockout HAP1 cell lysate (40 μg)
Lane 3 : HeLa cell lysate (40 μg)
Lane 4 : Jurkat cell lysate (40 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109535 observed at 48 kDa. Red - loading control, ab8245, observed at 37 kDa.

Unpurified ab109535 was shown to specifically react with TDP43 when TDP43 knockout samples were used. Wild-type and TDP43 knockout samples were subjected to SDS-PAGE. ab109535 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10,000 dilution respectively and incubated overnight at 4C. Blots were developed with IRDye^® 800CW Goat anti-Rabbit IgG (H + L) and IRDye^® 680 Goat anti-Mouse IgG (H + L) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-TDP43 antibody [EPR5810] - BSA and Azide free (ab185133)

Predicted band size: 44 kDa

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