- MA1-19731 - Invitrogen Antibodies DAXX antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blotting analysis of Daxx expression in human U937 cell line with anti-Daxx (DAXX-03) purified Monoclonal antibody (Product # MA1-19731).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of DAXX was achieved by transfecting SH-SY5Y cells with DAXX specific siRNAs (Silencer® select Product # s3935, s3936). Western blot analysis (Fig. a) was performed using whole cell extracts from the SH-SY5Y knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with DAXX Monoclonal Antibody (DAXX-03) (Product # MA1-19731, 1 µg/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 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 DAXX.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-DAXX Monoclonal Antibody (DAXX-03) (Product # MA1-19731) and a 100kDa band corresponding to DAXX was observed in all cell lysates tested. Whole cell lysates (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2), K-562 (Lane 3), MCF7 (Lane 4), U-2 OS (Lane 5) and SH-SY5Y (Lane 6) were electrophoresed using Novex® 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 (2µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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 DAXX 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 DAXX Monoclonal Antibody (DAXX-03) (Product # MA1-19731) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766) 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 strong nuclear and faint cytoplasmic localization. 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: Chromatin Immunoprecipitation (ChIP) assay of endogenous DAXX protein using Anti-DAXX Antibody: ChIP was performed using Anti-DAXX Monoclonal Antibody (Product # MA1-19731) 5 µg, on sheared chromatin from Camptothecin treated HeLa cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Mouse IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to promoter of PSMD12, TGM2, NDUFV1, KLK3 and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA1-19731