- 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: 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 (Heavy Chain) 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: 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 (Heavy Chain) 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.

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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