- MA5-15028 - Invitrogen Antibodies NEDD8 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NEDD8 was achieved by transfecting HeLa with NEDD8 specific siRNAs (Silencer® select Product # s226684, s9424). Western blot analysis (Fig. a) was performed using whole cell extracts from the NEDD8 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NEDD8 Monoclonal Antibody (B.226.2) (Product # MA5-15028, 1:1000 dilution) 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 NEDD8.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using anti-NEDD8 Monoclonal Antibody (B.226.2) (Product # MA5-15028) and a 9 kDa band corresponding to NEDD8 was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2), Jurkat (Lane 3), PC-12 (Lane 4), THP-1 (Lane 5), K-562 (Lane 6), HL-60 (Lane 7), Raw 264.7 (Lane 8), tissue extracts of Rat testis (Lane 9), Mouse brain (Lane 10) and Mouse colon (Lane 11) were electrophoresed Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX). 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 NEDD8 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 NEDD8 Monoclonal Antibody (B.226.2) (Product # MA5-15028) at 1:100 dilution 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 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 localization to the cytoplasm and nucleus. 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 analysis of NEDD8 in paraffin-embedded human colon carcinoma using a NEDD8 monoclonal antibody (Product # MA5-15028).

MA5-15028