- PA5-19508 - Invitrogen Antibodies NCL antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of A431 Whole Cell Lysate using Product # PA5-19508, Nucleolin primary antibody at a dilution of 1 µg/mL. Blot treated with a secondary IR Dye680-conjugated Goat polyclonal anti-Rabbit antibody was used at a dilution of 1:10000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Nucleolin (NCL) was achieved by transfecting HeLa with Nucleolin specific siRNAs (Silencer® select Product # s9314). Western blot analysis (Fig. a) was performed using Whole Cell Lysate extracts from knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Nucleolin Polyclonal Antibody (Product # PA5-19508, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of both the bands has been done together, this western blot is shown in histogram (Fig. b). Decrease in signal of all the bands upon siRNA mediated knock down confirms that antibody is specific to Nucleolin. Nucleolin is a ubiquitinylated protein, for which the molecular weight is reported to be 76.6 kDa to 100 kDa, which can be observed clearly in the Fig. a.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Nucleolin Polyclonal Antibody (Product # PA5-19508). Nucleolin is a ubiquitinylated protein for which the molecular weight is reported to be 76.6 kDa to 100 kDa, which can be observed in the cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), SH-SY5Y (Lane 2), NTERA-2 cl.D1 (Lane 3), A-431 (Lane 4), PANC-1 (Lane 5), Raji (Lane 6), Mouse liver (Lane 7) and Mouse brain (Lane 8) were electrophoresed using Novex® 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 (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)
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Experimental details: Immunofluorescence analysis of Nucleolin was performed using 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 Nucleolin Polyclonal Antibody (Product # PA5-19508) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 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 nuclear expression of Nucleolin. 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 (formalin-fixed, paraffin-embedded) staining of Human Tonsil tissue using Product # PA5-19508, anti-Nucleolin antibody. Primary antibody was used at a concentration of 1 µg/mL and exposed for 15 mins at room temp. The sample was pretreated using heat mediated antigen retrieval with Sodium Citrate Buffer (pH6/20mins). The detection method was a HRP conjugated polymer, DAB chromogen and the sample was counterstained with haematoxylin and mounted with DPX.

PA5-19508