- 703741 - Invitrogen Antibodies TDRD3 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-TDRD3 Recombinant Rabbit Monoclonal Antibody (Product # 703741) and a 140 kDa band corresponding to TDRD3 was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1) and MDA-MB-231 (Lane 2) were electrophoresed using Novex® NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:400 dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # A27036, 1:7000 dilution) using the iBright FL 1500 (Product # A44115). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TDRD3 was achieved by transfecting SH-SY5Y cells with TDRD3 specific siRNA (Silencer® select Product # s37606 & s37605). Western blot analysis (Fig a) was performed using whole cell extracts from TDRD3 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-TDRD3 Recombinant Rabbit Monoclonal Antibody (Product # 703741, 1:400 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:7000 dilution). Densitometric analysis of this western blot is shown in the histogram (Fig b). Loss of signal upon siRNA mediated knockdown confirms that antibody is specific to TDRD3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: For immunofluorescence analysis, SH-SY5Y cells were fixed and permeabilized for detection of endogenous TDRD3 using Anti-TDRD3 Recombinant Rabbit Monoclonal Antibody (Product # 703741, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731, 1:2000). Panel a) shows representative cells that were stained for detection and localization of TDRD3 protein (green), Panel b) is stained for nuclei (blue) using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic and nuclear localization of TDRD3. 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)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay for endogenous TDRD3 protein using TDRD3 Antibody: ChIP was performed with the MAGnify ChIP System kit (Product # 49-2024) using TDRD3 Recombinant Rabbit Monoclonal Antibody (Product # 703741, 5 µg) on sheared chromatin from 2 million SH-SY5Y cells. Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to the promoter regions of DNAJC9, NANOG, NRAS, gene bodies of TRIAP1, EGR1 and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

703741