- 701079 - Invitrogen Antibodies RELA antibody

Submitted references Hemoglobin oxidation generates globin-derived peptides in atherosclerotic lesions and intraventricular hemorrhage of the brain, provoking endothelial dysfunction.

Posta N, Csősz É, Oros M, Pethő D, Potor L, Kalló G, Hendrik Z, Sikura KÉ, Méhes G, Tóth C, Posta J, Balla G, Balla J
Laboratory investigation; a journal of technical methods and pathology 2020 Jul;100(7):986-1002

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HT-1080 (Lane 1), THP-1 (Lane 2), A431 (Lane 3), MCF7 (lane 4) and HEK-293 (lane 5). The blots were probed with Anti-NFkB p65 Recombinant Rabbit Monoclonal Antibody (Product # 701079, 2-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 60 kDa band corresponding to NFkB p65 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NFkB p65 was achieved by transfecting HeLa with NFkB p65 specific siRNAs (Silencer® select Product # s11916, s11914). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the NFkB p65 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NFkB p65 Recombinant Rabbit Monoclonal Antibody (4-2H22L23) (Product # 701079, 0.5 µg/mL concentration) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000 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 NFkB p65.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NFkB p65 in whole cell extract (50 µg/lane) from Hela cells (lane 1) and K562 cells (lane 2) using a NFkB p65 recombinant rabbit monoclonal antibody (Product # 701079) at a dilution of 2.5 µg/mL. Detection was performed using an HRP-conjugated goat anti-rabbit secondary antibody at a dilution of 1:2000 followed by chemiluminescence (ECL). Results show a band at ~60 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NFkB p65 was performed using 70% confluent log phase HeLa treated with human TNF-alpha (20 ng/mL, 40 minutes). The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with NFkB p65 Recombinant Rabbit Monoclonal Antibody (4-2H22L23) (Product # 701079) at 5 µg/mL concentration 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 translocation from cytoplasm upon treatment. Panel e represents cytoplasmic localization in untreated cells. Panel f 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: Immunohistochemistry analysis of NFKB P65 showing staining in the cytoplasm of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a NFKB P65 Recombinant Rabbit Monoclonal Antibody (Product # 701079) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of NFkB p65 was done on A549 cells treated with TNF alpha (50ng/mL, 20 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with ABfinity™ NFkB p65 Recombinant Rabbit Monoclonal Antibody (701079, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Enrichment of endogenous NF-kappa B p65 protein at specific gene loci using Anti-NF-kappa B p65 Recombinant Rabbit Monoclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-NF-kappa B p65 Recombinant Rabbit Monoclonal Antibody (Product # 701079, 5 µg) on sheared chromatin from 2 million HeLa cells treated with 50 ng/mL of TNFalpha for 45 minutes 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 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoter of active IL-8, IkB gene, used as positive control target, and the SAT2, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

701079

Antibody data