- PA5-30654 - Invitrogen Antibodies VPS35 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using VPS35 Polyclonal Antibody (Product # PA5-30654). Sample (30 µg of whole cell lysate). Lane A: HCT116. 7.5% SDS PAGE. VPS35 Polyclonal Antibody (Product # PA5-30654) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of VPS35 was performed by loading 20 µg of HAP1 WT (lane 1) and VPS35 KO (lane 2) cell lysates in RIPA buffer onto a 4-15% gradient polyacrylamide gel. 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. VPS35 was detected at approximately 91 kDa using VPS35 Polyclonal Antibody (Product # PA5-30654) at a dilution of 1:1000 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)
Main image
Experimental details: Western Blot using VPS35 Polyclonal Antibody (Product # PA5-30654). Sample (30 µg of whole cell lysate). Lane A: NIH-3T3. Lane B: JC. 7.5% SDS PAGE. VPS35 Polyclonal Antibody (Product # PA5-30654) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of VPS35 was achieved by transfecting HeLa with VPS35 specific siRNAs (Silencer® select Product # s31375, s31376). Western blot analysis (Fig. a) was performed using whole cell extracts from the VPS35 KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with VPS35 Polyclonal Antibody (Product # PA5-18248, 1:1000 dilution) and Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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 VPS35. .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-VPS35 Rabbit Polyclonal Antibody (Product # PA5-30654) and a 80 kDa band corresponding to VPS35 was observed across the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), A549 (Lane 2), U-937 (Lane 3), SH-SY5Y (Lane 4), HeLa (Lane 5), tissue extracts of Mouse Lung (Lane 6) and Rat Lung (Lane 7) were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0322BOX). 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 (1:1000 Dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). 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: Immunohistochemical analysis of paraffin-embedded A549 xenograft, using VPS35 (Product # PA5-30654) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of VPS35 was performed in HeLa whole cell extracts using 5 µg of VPS35 Polyclonal Antibody (Product # PA5-30654). Samples were transferred to a membrane and probed with VPS35 Polyclonal Antibody as a primary antibody and an HRP-conjugated anti-Rabbit IgG was used as a secondary antibody.

PA5-30654