- 711051 - Invitrogen Antibodies TARDBP antibody

Submitted references TDP-43 as structure-based biomarker in amyotrophic lateral sclerosis.

Beyer L, Günther R, Koch JC, Klebe S, Hagenacker T, Lingor P, Biesalski AS, Hermann A, Nabers A, Gold R, Tönges L, Gerwert K
Annals of clinical and translational neurology 2021 Jan;8(1):271-277

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Nuclear Enriched extracts (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), Caco-2 (Lane 3), A549 (Lane 4) and SH-SY5Y (Lane 5). The blots were probed with Anti-TDP43 Recombinant Rabbit Polyclonal Antibody (Product # 711051, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 45 kDa bands corresponding to TDP43 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing of TARDBP was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042) (Assay ID CRISPR1087594_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of TARDBP was performed by loading 30 µg of HeLa CAS9 (Lane 1) and HeLa CAS9 cells transduced with TARDBP Lentiviral sgRNA (Lane 2) whole cell extracts. The blot was probed with Anti-TDP-43 Recombinant Polyclonal Antibody (1HCLC)(Product # 711051) using 2 µg/mL dilution and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific to TARDBP (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of TDP-43 was performed by loading 50 µg of WT (lane 1) and TARDBP CRISPR KO (lane 2) HAP1 cell lysates in RIPA buffer onto a 5-16% gradient polyacrylamide gel. Proteins on the blots were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. TDP-43 was detected at approximately 45 kDa using a TDP-43 recombinant polyclonal antibody (Product # 711051) at a dilution of 1:1000 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4 deg. The peroxidase-conjugated secondary antibody (Product # 65-6120) was diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106). Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TDP-43 was achieved by transfecting HeLa with HOMER1 specific siRNAs (Silencer® select Product # S23829, S23830). Western blot analysis (Fig. a) was performed using whole cell lysate from the TDP-43 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TDP-43 Recombinant Polyclonal Antibody (1HCLC) (Product # 711051, 1ug/ml dilution) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to TDP-43.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, A549 cells were fixed and permeabilized for detection of endogenous TDP43 using Anti- TDP43 Recombinant Rabbit Polyclonal Antibody (Product # 711051, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of TDP43 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of TDP43. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence of TDP-43 was performed using HAP1 WT and TARDBP KO cells that were transfected with a green or a far-red fluorescent dye, respectively. Post-transfection, WT and KO cells were mixed and plated to a 1:1 ratio on glass coverslips as a mosaic and incubated for 24 hrs. Cells were fixed in 4% PFA (in PBS) for 15 min at RT; cells were permeabilized with 0.1% Triton X-100 for 10 min at RT and blocked with PBS with 5% BSA, 5% goat serum, and 0.01% Triton X-100 for 30 min. Cells were stained with TDP-43 Recombinant Polyclonal Antibody (1HCLC) (Product # 711051) at a 1:500 dilution overnight at 4 degree celcius. Secondary antibody incubation was performed using 1 µg/mL of Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (Product # A21429) together with DAPI for 1 hr at RT. Imaging was performed with a 20X water immersion objective. Representative images where WT and KO cells are outlined with a green (WT) or magenta (KO) line, respectively, are shown. The top and bottom panels show antibody and DAPI stainings, respectively. Scale bar = 10 μm. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, Hela cells were fixed and permeabilized for detection of endogenous TDP43 using Anti- TDP43 Recombinant Rabbit Polyclonal Antibody (Product # 711051, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of TDP43 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of TDP43. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of TDP-43 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with TDP-43 Recombinant Polyclonal Antibody (1HCLC) (Product # 711051) at 2 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: RNA immunoprecipitation (RIP) western of TARDBP was performed on K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 µg of TARDBP Recombinant Rabbit Polyclonal Antibody (Product # 711051) rotating 60 min at RT. The immune complexes were captured on 625 µg of Anti- rabbit coated Dynabeads (Product # 11204D), washed extensively, and eluted with NuPAGE™ LDS Sample Buffer (Product # NP0007). Samples were resolved onto NuPAGE™ 4-12% Bis-Tris gel (Product # NP0335BOX). Lanes 1 and 3 are input and lanes 2 and 4 are IP. Proteins were transferred to PVDF membrane (Product # IB23001). Membrane was blocked in 5% milk. Target was detected using a TARDBP Recombinant Rabbit Polyclonal Antibody (Product # 711051) at a dilution of 1:2000, followed by a 1:4000 dilution of secondary antibody. Chemiluminescent detection was performed using ECL Western blotting Substrate (Product # 32106). Data courtesy of the Yeo lab as part of the ENCODE project (www.encodeproject.org).

711051

Antibody data