- PA5-22294 - Invitrogen Antibodies TRIM21 antibody

Katsiougiannis S, Tenta R, Skopouli FN
Clinical and experimental immunology 2015 Aug;181(2):244-52

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of SSA1 using 30 µg of MOLT4 lysate. Samples were loaded onto a 10% SDS-PAGE gel and probed with a SSA1 polyclonal antibody (Product # PA5-22294) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of TRIM21 was performed by separating 30 µg of non-transfected (–) and transfected (+) 293T whole cell extracts by 10% SDS-PAGE. Proteins were transferred to a membrane and probed with a TRIM21 Polyclonal Antibody (Product # PA5-22294) at a dilution of 1:1000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of TRIM21 was achieved by transfecting HeLa cells with TRIM21 specific siRNAs (Silencer® select Product # S13463, S13464) followed by treatment with IFNa (10 ng/mL for 16 hours). Western blot analysis (Fig. a) was performed using whole cell extracts from the TRIM21 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TRIM21 Polyclonal Antibody (Product # PA5-22294, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 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 TRIM21.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using TRIM21 Polyclonal Antibody (Product # PA5-22294) and a 54 kDa band corresponding to TRIM21 was observed across all cell lines tested and was observed to be upregulated upon treatment of HeLa cells with IFNa and HCT116 with cisplatin. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with with IFNa (10 ng/mL, 16 hours) (Lane 2), HCT 116 (Lane 3), HCT 116 treated with with cisplatin (3.75 µg/mL, 24 hours) (Lane 4), HT-29 (Lane 5) and RT-4 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX), 10 well. 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 with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580). Upregulation of TRIM21 expression upon cell treatment demonstrates specificity of the antibody. Some uncharacterized bands were observed between 15-20 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of SSA1 in paraformaldehyde-fixed A431 cells using a SSA1 polyclonal antibody (Product # PA5-22294) at a 1:200 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of TRIM21 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 TRIM21 Polyclonal Antibody (Product # PA5-22294) 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). Panel d represents the merged image showing nuclear and cytoplasmic 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 analysis of paraffin-embedded DLD-1 xenograft , using SSA1 (Product # PA5-22294) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of SSA1 was performed in 293T whole cell extracts using 5 µg of TRIM21 Polyclonal Antibody (Product # PA5-22294). Samples were transferred to a membrane and probed with TRIM21 Polyclonal Antibody as a primary antibody and an HRP-conjugated anti-Rabbit IgG was used as a secondary antibody.

PA5-22294