- PA5-19974 - Invitrogen Antibodies DIABLO antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot Validation in Human Heart Tissue Lysate. Loading: 15 µg of lysates per lane. Antibodies: DIABLO Polyclonal Antibody (Product # PA5-19974) (1 µg/mL), 1h incubation at RT in 0.05 NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10,000 dilution. (A) Without blocking peptide (B) With blocking peptide

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot Validation in Human Heart Tissue Lysate. Loading: 15 µg of lysates per lane. Antibodies: DIABLO Polyclonal Antibody (Product # PA5-19974) (1 µg/mL), 1h incubation at RT in 0.05 NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10,000 dilution. (A) Without blocking peptide (B) With blocking peptide

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot Validation in Human, Mouse and Rat Cell Lines. Loading: 15 µg of lysates per lane. Antibodies: DIABLO Polyclonal Antibody (Product # PA5-19974) (1 µg/mL), 1h incubation at RT in 0.05 NFM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10,000 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot Validation in Mouse 3T3/NIH Cells. Loading: 15 µg of lysate. Antibodies: DIABLO Polyclonal Antibody (Product # PA5-19974) (1 µg/mL), 1h incubation at RT in 0.05 NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10,000 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of DIABLO was achieved by transfecting HeLa with DIABLO specific siRNAs (Silencer® select Product # S32187, S32188). Western blot analysis (Fig. a) was performed using Whole cell extracts from the DIABLO knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with DIABLO Polyclonal Antibody (Product # PA5-19974, at a concentration of 1 ug/ml) 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 DIABLO.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-DIABLO Polyclonal Antibody(Product # PA5-19974) and a 21kDa band corresponding to DIABLO was observed across the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), MCF-7 (Lane 2), Hep G2 (Lane 3) and Mouse Testis (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 ug/ml) 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 DIABLO 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 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with DIABLO Polyclonal Antibody (Product # PA5-19974) 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), (1:2000 dilution), 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 dilution). Panel d represents the merged image showing Mitochondrial localization. Panel e represents control HeLa 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: Immunohistochemical analysis of paraffin-embedded Human Ovary tissue using DIABLO Polyclonal Antibody (Product # PA5-19974) at 5 µg/mL. Tissue was fixed with formaldehyde and blocked with 0.1 serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

PA5-19974