- PA5-67313 - Invitrogen Antibodies ORC2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of ORC2 in human cell line RT-4 and human cell line U-251 MG. Samples were probed using an ORC2 Polyclonal Antibody (Product # PA5-67313).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of ORC2 was achieved by transfecting HeLa cells with ORC2 specific siRNAs (Silencer® select Product # s9896, s9895). Western blot analysis (Fig. a) was performed using whole cell extracts from the HeLa knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with ORC2 Rabbit Polyclonal Antibody (Product # PA5-67313, 1:500 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 ORC2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-ORC2 Monoclonal Antibody (Product # PA5-67313) and a ~66 kDa band was observed across the panel tested. Modified whole cell extracts (1% SDS) (50 µg lysate) of A549 (Lane 1), PC-3 (Lane 2), HeLa (Lane 3), HCT 116 (Lane 4) and Raji (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:500 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 staining of ORC2 in human cell line Hep G2 shows localization to nucleoplasm. Samples were probed using an ORC2 Polyclonal Antibody (Product # PA5-67313).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of ORC2 was performed using 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 ORC2 Monoclonal Antibody (Product # PA5-67313) at 1:200 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at 1:2000 dilution for 45 minutes at room temperature (Panel a: green) in HeLa cells. 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 of HeLa cells showing nuclear localization for ORC2. 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: Chromatin Immunoprecipitation (ChIP) assay of endogenous ORC2 protein using ORC2 Antibody: ChIP was performed using Anti-ORC2 Rabbit Polyclonal Antibody (Product # PA5-67313, 2.5 µg) on sheared chromatin from HeLa cells 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 promoter region of GAPDH, transcription 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.

PA5-67313