- PA5-27854 - Invitrogen Antibodies NRF1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NRF1 Polyclonal Antibody detects NRF1 protein by western blot analysis. A. 30 µg 293T whole cell lysate/extract. B. 30 µg 293T nuclear lysate/extract. C. 30 µg A431 whole cell lysate/extract . D. 30 µg A431 nuclear lysate/extract. E. 30 µg HeLa whole cell lysate/extract. F. 30 µg HeLa nuclear lysate/extract. G. 30 µg HepG2 whole cell lysate/extract. H. 30 µg HepG2 nuclear lysate/extract.10 % SDS-PAGE. NRF1 Polyclonal Antibody (Product # PA5-27854) dilution: 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: KD of NRF1 was achieved by transfecting HeLa cells with NRF1 specific siRNAs (Silencer® select Product # s32459, s32460). Western blot analysis (Fig. a) was performed using Whole cell extracts from the NRF1 KD cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NRF1 Polyclonal Antibody (Product # PA5-27854, 1:2000 dilution) and Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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 NRF1. .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-NRF1 Rabbit Polyclonal Antibody (Product # PA5-27854). A 59 kDa band corresponding to NRF1 and a 100 kDa uncharacterized band (*) were observed across cell lines tested. Modified whole cell extracts (1% SDS) (30 µg lysate) of HeLa (Lane 1), Hep G2 (Lane 2), PC-3 (Lane 3), A-431 (Lane 4), U-2 OS (Lane 5) and A549 (Lane 6) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris 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 Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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: Immunocytochemistry-Immunofluorescence analysis of NRF1 was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: NRF1 Polyclonal Antibody (Product # PA5-27854) diluted at 1:500. Blue: Hoechst 33342 staining. Scale bar = 10 µm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NRF1 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 1% BSA for 1 hour at room temperature. The cells were labeled with NRF1 Rabbit Polyclonal Antibody (Product # PA5-27854) at 1:250 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (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, 1:300). Panel d represents the composite image showing nuclear localization of NRF1. 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: NRF1 Polyclonal Antibody detects NRF1 protein at nucleus on human lung adenocarcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human lung adenocarcinoma. NRF1 Polyclonal Antibody (Product # PA5-27854) diluted at 1:250. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NRF1 Polyclonal Antibody detects NRF1 protein at nucleus on mouse colon by immunohistochemical analysis. Sample: Paraffin-embedded mouse colon. NRF1 Polyclonal Antibody (Product # PA5-27854) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NRF1 Polyclonal Antibody detects NRF1 protein at nucleus on mouse colon by immunohistochemical analysis. Sample: Paraffin-embedded mouse colon. NRF1 Polyclonal Antibody (Product # PA5-27854) dilution: 1:500. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NRF1 Polyclonal Antibody immunoprecipitates NRF1 protein in IP experiments. IP samples: 293T whole cell extract. A. Control with 4 µg of preimmune Rabbit IgG. B. Immunoprecipitation of NRF1 protein by 4 µg NRF1 Polyclonal Antibody (Product # PA5-27854). 10 % SDS-PAGE. The immunoprecipitated NRF1 protein was detected by NRF1 Polyclonal Antibody (Product # PA5-27854) diluted at 1:1,000.

PA5-27854