- 703745 - Invitrogen Antibodies WDR5 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of WDR5 was achieved by transfecting HCT116 cells with WDR5 specific siRNA (Silencer® select Product # s21862 & s21863). Western blot analysis (Fig a) was performed using modified whole cell extracts (1% SDS) from WDR5 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-WDR5 Recombinant Rabbit Monoclonal Antibody (Product # 703475, 1:5000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:5000 dilution). Densitometric analysis of this western blot is shown in the histogram (Fig b). Loss of signal upon siRNA mediated knockdown confirms that antibody is specific to WDR5.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-WDR5 Recombinant Rabbit Monoclonal Antibody (Product # 703475) and a 37 kDa band corresponding to WDR5 was observed across the cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of HCT 116 (Lane 1), HT-29 (Lane 2), SW480 (Lane 3), COLO 205 (Lane 4), THP-1 (Lane 5) and PC-12 (Lane 6) were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris 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 (1:5000 dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:5000 dilution). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, HCT 116 cells were fixed and permeabilized for detection of endogenous WDR5 using Anti-WDR5 Recombinant Rabbit Monoclonal Antibody (Product # 703475, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731, 1:2000). Panel a) shows representative cells that were stained for detection and localization of WDR5 protein (green), Panel b) is stained for nuclei (blue) using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of WDR5. 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)
Main image
Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous WDR5 protein using Anti-WDR5 Antibody: ChIP was performed using Anti-WDR5 Recombinant Rabbit monoclonal Antibody (Product # 703745, 5 µg) on sheared chromatin from 2 million HCT116 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to CDKN1A intron1, c-MYC promoter, Gene body (+2Kb) and transcriptional start site of GAPDH and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

703745