- 702788 - Invitrogen Antibodies ATXN3 antibody

Submitted references Mitochondrial Dysfunction in Spinocerebellar Ataxia Type 3 Is Linked to VDAC1 Deubiquitination.

Harmuth T, Weber JJ, Zimmer AJ, Sowa AS, Schmidt J, Fitzgerald JC, Schöls L, Riess O, Hübener-Schmid J
International journal of molecular sciences 2022 May 25;23(11)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Ataxin 3 was achieved by transfecting U87MG cells with Ataxin 3 specific siRNA (Silencer® select Product # s230538 and Product # s8794). Western blot analysis (Fig a) was performed using whole cell extract from the Ataxin 3 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-Ataxin 3 Recombinant Rabbit Monoclonal Antibody (Product # 702788, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to Ataxin 3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1), U-87 MG (Lane 2), HeLa (Lane 3), Neuro-2a (Lane 4) and tissue extracts (30 µg lysate) of Mouse Brain (Lane 5). The blots were probed with Anti-Ataxin 3 Recombinant Rabbit Monoclonal Antibody (Product # 702788, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). 42 and 40 kDa bands corresponding to Ataxin-3 was observed across the cell lines and tissue tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), 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)
Main image
Experimental details: Western blot analysis of Ataxin 3 was performed by loading 30 µg of HEK293T wildtype (Lane 1), HEK293T Ataxin 3 knockout (Lane 2) whole cell extracts. The blot was probed with Anti-Ataxin 3 Monoclonal Antibody (Product # 702788) (2.5 µg/ml) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036) (1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to Ataxin 3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of Ataxin-3 was performed by loading 30 µg of WT (lane 1) and ATXN3 CRISPR KO (lane 2) HEK293T 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. ATXN3 was detected at approximately 42 kD using an ATXN3 recombinant monoclonal antibody (Product # 702788) at a dilution of 1:50 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4°C. 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)
Main image
Experimental details: For immunofluorescence analysis, SH-SY5Y cells were fixed and permeabilized for detection of endogenous Ataxin 3 using Anti-Ataxin 3 Recombinant Rabbit Monoclonal Antibody (Product # 702788, 5 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 Ataxin 3 (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 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 Ataxin 3. 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: Knockdown of Ataxin 3 was achieved by transfecting PC-3 cells with specific siRNA (Silencer® select Product # s230538 + s8794). Immunofluorescence analysis was performed on PC-3 cells (untransfected, panel a-d), transfected with Ataxin 3 specific siRNA (panel i-l) or non-specific scrambled siRNA (panels e-h). Cells were fixed, permeabilized, and labelled with Anti-Ataxin 3 Recombinant Rabbit Monoclonal Antibody (Product # 702788, 1:100 dilution), followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Significant reduction of signal was observed upon siRNA mediated knockdown (panel i-l) confirming specificity of the antibody to Ataxin 3 (green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Ataxin-3 was performed on HEK293T cell lysates. Antibody-bead conjugates were prepared by adding 1 µg of ATXN3 recombinant monoclonal antibody (Product # 702788) with 30 µL of protein A-Sepharose beads and rocked overnight at 4°C. 1 mg of lysate was incubated with an antibody-bead conjugate for 2 hours at 4°C. Following centrifugation and multiple washes, 10% starting material (SM), 10% unbound fraction (UB) and immunoprecipitated fraction (IP) were processed for immunoblot using another ATXN3 polyclonal antibody. Ponceau stained transfer of blot is shown. Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.

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