- PA5-52602 - Invitrogen Antibodies RAD18 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of RAD18 in control (vector only transfected HEK293T lysate) and RAD18 over-expression lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells). Samples were probed using a RAD18 Polyclonal Antibody (Product # PA5-52602).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of RAD18 was achieved by transfecting HeLa with RAD18 specific siRNAs (Silencer® select Product # s32295 and s32296). Western blot analysis (Fig. a) was performed using Whole Cell Extract-WCL from the RAD18 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RAD18 Polyclonal Antibody (Product # PA5-52602, 0.3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). 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 RAD18.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-RAD18 Polyclonal Antibody(Product # PA5-52602) and a 75 kDa and 80kDa band corresponding to RAD18 were observed across cell lines tested. This product does not show reactivity to mouse tissues. Whole Cell Extract-WCL (30 µg lysate) of HeLa (Lane 1), HCT 116 (Lane 2), U-87 MG (Lane 3), A549 (Lane 4), PC-3 (Lane 5), Mouse Heart (Lane 6), Mouse Brain (Lane 7), Mouse Liver (Lane 8) and Mouse Adipose (Lane 9) 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 (0.4 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) 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 staining of RAD18 in human cell line A-431 shows positivity in nucleus but excluded from the nucleoli. Samples were probed using a RAD18 Polyclonal Antibody (Product # PA5-52602).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of RAD18 in human cell line A-431 using a RAD18 Polyclonal Antibody (Product # PA5-52602) shows localization to nucleoplasm and nuclear bodies.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of RAD18 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 RAD18 Polyclonal Antibody (Product # PA5-52602) at 2 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (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 nuclear 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: Immunohistochemical staining of RAD18 in human lymph node shows distinct nuclear positivity in germinal center cells. Samples were probed using a RAD18 Polyclonal Antibody (Product # PA5-52602).

PA5-52602