- PA5-29795 - Invitrogen Antibodies CYLD antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Cylindromatosis 1 using 30 µg of HeLa lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a Cylindromatosis 1 polyclonal antibody (Product # PA5-29795) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of CYLD was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a CYLD Polyclonal Antibody (Product # PA5-29795) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of CYLD was achieved by transfecting U-87 MG with CYLD specific siRNAs (Silencer® select Product # S590). Western blot analysis (Fig. a) was performed using whole cell extracts from the U-87 MG knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with CYLD Polyclonal Antibody (Product # PA5-29795, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A27036, 0.25µg/ml, 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 CYLD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-CYLD Polyclonal Antibody (Product # PA5-29795) and a ~110 kDa band corresponding to CYLD was observed across all the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of U-87 MG (Lane 1), HeLa (Lane 2), Raji (Lane 3), tissue extracts of Mouse Brain (Lane 4) and Mouse Lungs (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 (1:2000 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: Immunofluorescent analysis of Cylindromatosis 1 in methanol-fixed HeLa cells using a Cylindromatosis 1 polyclonal antibody (Product # PA5-29795) at a 1:200 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of CYLD was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 100% Acetone for 5 minutes, at -20 degree Celsius and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with CYLD Polyclonal Antibody (Product # PA5-29795) at 1:100 dilution 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) 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 localization to the cell membrane. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-29795