- PA5-28922 - Invitrogen Antibodies DKC1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using DKC1 Polyclonal Antibody (Product # PA5-28922). Sample (30 µg of whole cell lysate). Lane A: HeLa. Lane B: HepG2 . 7.5% SDS PAGE. DKC1 Polyclonal Antibody (Product # PA5-28922) diluted at 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of DKC1 was achieved by transfecting Hep G2 with DKC1 specific (Silencer® select Product # s4110, s4111). Western blot analysis (Fig. a) was performed using Modified whole cell extracts (1% SDS) from the untransfected cells (Lane 1), non-specific scrambled siRNA transfected cells (Lane 2) and DKC1 knockdown cells (Lane 3). The blot was probed with DKC1 Polyclonal Antibody (Product # PA5-28922, 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). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to DKC1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using DKC1 Polyclonal Antibody (Product # PA5-28922) and a 57 kDa band corresponding to DKC1 was observed in cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of MCF7 (Lane 1) and Hep G2 (Lane 2) were electrophoresed using 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 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 analysis of DKC1 in paraformaldehyde-fixed HeLa cells using a DKC1 polyclonal antibody (Product # PA5-28922) (Green) at a 1:500 dilution. Alpha-tubulin filaments were labeled with Product # PA5-29281 (Red) at a 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of DKC1 was performed using 70% confluent log phase Hep G2 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 DKC1 Polyclonal Antibody (Product # PA-28922) 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 (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 nucleus. Panel f 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 paraffin-embedded BT474 xenograft, using DKC1 (Product # PA5-28922) antibody at 1:250 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-28922