- PA5-78203 - Invitrogen Antibodies TOP1 antibody

No submitted references: Submit reference

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of TOP2A in 293T cells using 5 µg of protein. Samples were separated with 7.5% SDS-PAGE and incubated with TOP2A polyclonal antibody (Product # PA5-78203) using a dilution of 1:2000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of TOP1 was performed by separating 50 µg of Rat tissue extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a TOP1 Polyclonal Antibody (Product # PA5-78203) at a dilution of 1:500. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Topo I antibody detects Topo I protein by western blot analysis. 293T whole cell extracts and nuclear extracts (5 µg) were separated by 7.5% SDS-PAGE, and the membrane was blotted with Topo I antibody TOP1 Polyclonal Antibody (Product # PA5-78203) at a dilution of 1:2,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of TOP1 was achieved by transfecting HEK-293 with TOP1 specific siRNAs (Silencer® select Product # s14305 ). Western blot analysis (Fig. a) was performed using whole cell extracts from the TOP1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TOP1 Polyclonal Antibody (Product # PA5-78203, 1:2000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 TOP1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-TOP1 Polyclonal Antibody (Product # PA5-78203) and a 91 kDa band corresponding to TOP1 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1), MCF-7 (Lane 2), Hep G2 (Lane 3), Jurkat (Lane 4), NTERA-2 cl.D1 (Lane 5) and Caco-2 (Lane 6) 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)
Main image
Experimental details: Immunofluorescence analysis of TOP1 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 TOP1 Rabbit Polyclonal Antibody (Product # PA5-78203) at 5 µg/mL 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 (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). 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: Chromatin Immunoprecipitation (ChIP) assay of endogenous TOP1 protein using Anti-TOP1 Antibody: ChIP was performed using Anti-TOP1 Rabbit Polyclonal Antibody (Product # PA5-78203, 5 µg) on sheared chromatin from Camptothecin treated HCT 116 cells (10uM, 2hours) (using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to CDKN1A intron 1, 18s rDNA, promoter and transcriptional start site of GAPDH, and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

PA5-78203