- PA5-60717 - Invitrogen Antibodies SHARPIN antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of SHARPIN in Lane 1: Marker (kDa) 250, 130, 95, 72, 55, 36, 28, 17, 10; Lane 2: Human cell line RT-4; Lane 3: Human cell line U-251MG sp; Lane 4: Human plasma (IgG/HSA depleted); Lane 5: Human liver tissue. Samples were probed using a SHARPIN Polyclonal Antibody (Product # PA5-60717).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of SHARPin in human cell lines PC-3 and Caco-2 using a SHARPIN Polyclonal Antibody (Product # PA5-60717). Corresponding SHARPIN RNA-seq data are presented for the same cell lines. Loading control: Anti-HSP90B1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of SHARPIN was achieved by transfecting A549 with SHARPIN specific siRNAs (Silencer® select Product # s195427, s37848). Western blot analysis (Fig. a) was performed using Whole cell extracts from the SHARPIN knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with SHARPIN Polyclonal Antibody (Product # PA5-60717, 0.1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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 SHARPIN.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SHARPIN Polyclonal Antibody (Product # PA5-60717) and a 42 kDa band corresponding to SHARPIN was observed across cell lines tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), MCF7 (Lane 2), HeLa (Lane 3), PC-3 (Lane 4), THP-1 (Lane 5) and Jurkat (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 (0.1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (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: Immunofluorescent staining of SHARPIN in human cell line MCF7 shows positivity in nucleus & cytoplasm. Samples were probed using a SHARPIN Polyclonal Antibody (Product # PA5-60717).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of SHARPIN was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with SHARPIN Polyclonal Antibody (Product # PA5-60717) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic(endosomal like pattern) localization. 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: Immunohistochemical staining of SHARPIN in human colon tissue shows strong cytoplasmic positivity in glandular cells. Samples were probed using a SHARPIN Polyclonal Antibody (Product # PA5-60717).

PA5-60717