- PA5-17652 - Invitrogen Antibodies RAN antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of RAN was achieved by transfecting HeLa with RAN specific siRNA (Silencer® select Product # s11769). Western blot analysis (Fig. a) was performed using whole cell extracts from the RAN knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RAN Polyclonal Antibody (Product # PA5-17652, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25µg/ml, 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 RAN.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-RAN Polyclonal Antibody (Product # PA5-17652) and a 28 kDa band corresponding to RAN was observed across all the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), HeLa (Lane 2), NIH/3T3 (Lane 3), Jurkat (Lane 4), MDA-MB-231 (Lane 5), MCF7 (Lane 6), NK-92 (Lane 7), tissue extracts of Mouse Testis (Lane 8) and Rat Testis (Lane 9) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 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)
Main image
Experimental details: Immunofluorescence analysis of RAN was performed using 70% confluent log phase HeLa 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 1 hour at room temperature. The cells were labeled with RAN Polyclonal Antibody (Product # PA5-17652) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing staining in nucleus. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-17652