- PA5-17080 - Invitrogen Antibodies ADAM9 antibody

Czarnek M, Stalińska K, Sarad K, Bereta J
Biology open 2022 Mar 15;11(3)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Disintegrin and metalloproteinase domain-containing protein 9 was achieved by transfecting U-87 MG cells with Disintegrin and metalloproteinase domain-containing protein 9 specific siRNAs (Silencer® select Product # S16684, S16686). Western Blot analysis (Fig. a) was performed using Whole cell extracts from the Disintegrin and metalloproteinase domain-containing protein 9 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with ADAM9 Polyclonal Antibody (Product # PA5-17080, 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 of the 73 kDa band of mature ADAM-9-L confirms that antibody is specific to Disintegrin and metalloproteinase domain-containing protein 9.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot was performed using Anti-ADAM9 Polyclonal Antibody (Product # PA5-17080) and a 90,73 kDa band corresponding to pro ADAM-9-L and the mature ADAM-9-L respectively was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), A-431 (Lane 2), Reh (Lane 3) and BJ (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 (1:1000 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 Disintegrin and metalloproteinase domain-containing protein 9 was performed using 70% confluent log phase A-431 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 45 minutes at room temperature. The cells were labeled with ADAM9 Polyclonal Antibody (Product # PA5-17080) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790, 1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415,1:300 dilution ). Panel d represents the merged image showing cytoplasm and secretory vesiclular like localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40x magnification in Cell Insight CX7 LZR instrument.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of ADAM9 from HeLa cell extracts using an ADAM9 polyclonal antibody (Product # PA5-17080) followed by Western blot analysis using the same antibody.

PA5-17080