- 702776 - Invitrogen Antibodies SPG11 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Spatacsin was achieved by transfecting HeLa cells with Spatacsin specific siRNA (Silencer® select Product # s37058+s37059). Western blot analysis was performed using membrane extract from the Spatacsin knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-Spatacsin Recombinant Rabbit Monoclonal Antibody (Product # 702776, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to Spatacsin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), SH-SY5Y (Lane 2) COS-7 (Lane 3) and U-87 MG (Lane 4). The blots were probed with Anti-Spatacsin Recombinant Rabbit Monoclonal Antibody (Product # 702776, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 278 kDa band corresponding to Spatacsin was observed across the cell lines 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 HiMark™ Pre-Stained Protein Standard (Product # LC5699). Resolved proteins were then transferred onto a nitrocellulose membrane by wet transfer method. 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: Western blot of SPG11 was performed by loading 30 µg of HAP1 WT (lane 1) and SPG11 KO (lane 2) cell lysates in RIPA buffer onto a NuPAGE™ 3 to 8%, Tris-Acetate, 1.0 mm, Midi Protein Gel (Product # WG1601BOX). Proteins on the blot were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. SPG11 was detected at approximately 279 kDa using SPG11 Recombinant Rabbit Monoclonal Antibody (2H25L11) (Product # 702776) at a dilution of 1:200 in 5% BSA in TBST overnight at 4 degree celsius. The blot was probed with Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Product # 65-6120) diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr at room temperature. Chemiluminescent detection was performed using ECL Western Blotting Substrate. 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: For immunofluorescence analysis, SH-SY5Y cells were fixed and permeabilized for detection of endogenous Spatacsin using Anti- Spatacsin Recombinant Rabbit Monoclonal Antibody (Product # 702776, 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 Spatacsin 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Spatacsin. Panel e) represents control cells without primary antibody to assess background. The images were captured at 60X magnification.

702776