- PA5-52331 - Invitrogen Antibodies RAD18 antibody

No submitted references: Submit reference

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
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 # PA5-52331, 1:1250) 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. Uncharacterized bands (*) at 35 kDa and 60 kDa were observed.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-RAD18 Polyclonal Antibody(Product # PA5-52331) and a 75 kDa and 80kDa band corresponding to RAD18 were observed across cell lines. Whole Cell Extract-WCL (30 µg lysate) of HeLa (Lane 1), HCT 116 (Lane 2), U-87 MG (Lane 3), A549 (Lane 4) and PC-3 (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 (1:1000) 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). Uncharacterized bands (*) at 35 kDa and 60 kDa were observed.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent staining of RAD18 in human cell line U-251 MG shows positivity in nucleus but excluded from the nucleoli. Samples were probed using a RAD18 Polyclonal Antibody (Product # PA5-52331).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
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 45 minutes at room temperature. The cells were labeled with RAD18 Polyclonal Antibody (Product # PA5-52331) at 1:100 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)
Main image
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 # PA5-52331).

PA5-52331