- PA5-54876 - Invitrogen Antibodies KIFAP3 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of KAP3 was achieved by transfecting SH-SY5Y with KAP3 specific siRNAs (Silencer® select Product # S22677, S22676). Western blot analysis (Fig. a) was performed using whole cell extracts from the KAP3 KD cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with KAP3 Polyclonal Antibody (Product # PA5-54876, 1:1000 dilution) and Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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 KAP3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-KAP3 Polyclonal Antibody (Product # PA5-54876) and a 80 kDa band corresponding to KAP3 was observed across cell lines and tissue tested. Whole cell extracts (30 µg lysate) of A-431 (Lane 1), NIH/3T3 (Lane 2), A549 (Lane 3), SH-SY5Y (Lane 4), MCF7 (Lane 5), DU 145 (Lane 6) and tissue extract (30 µg lysate) of Mouse Brain (Lane 7) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein 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 (1:1000 dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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 KAP3 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 KAP3 Polyclonal Antibody (Product # PA5-54876) at 5µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 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). Panel d represents the merged image showing staining in cytoskeleton. Panel e represents 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 staining of KAP3 in human adrenal gland tissue shows strong nuclear and cytoplasmic positivity in cortical cells. Samples were probed using a KAP3 Polyclonal Antibody (Product # PA5-54876).

PA5-54876