Invitrogen

NFkB p65 Polyclonal Antibody

Advanced Verification

IP-mass spectrometry View Details

6 Published Figures

11 References

View all (118) NFkB p65 antibodies

Datasheet

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Datasheet

Protocols

Questions & Answers

Tech Support

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NFkB p65 Antibody in Western Blot (WB) — Western blot analysis was performed on whole cell extracts (20 µg lysate) of NIH/3T3 (Lane 1), MCF7 (Lane 2), Hep G2 (Lane 3), Jurkat (lane 4) and HeLa (lane 5). The blots were probed with Anti-NF-kB p65 Rabbit Polyclonal Antibody (Product # PA1-186, 1-3 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 NF-kB p65 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), 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).

NFkB p65 Antibody in Immunoprecipitation (IP) — Immunoprecipitation of NF-kB p65 was performed on HeLa cells. Antigen-antibody complexes were formed by incubating 250 µg of HeLa whole cell lysate with 2.5 µg of an NF-kB p65 polyclonal antibody (Product # PA1-186) overnight on an end-over-end mixer at 4C. The immune complexes were captured on 100 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane (Product # 88518) using the G2 Fast Blotter (Product # 62288), and blocked with StartingBlock T20 (TBS) Blocking Buffer (Product # 37543) for at least 1 hour at room temperature. The membrane was probed with an NF-kB p65 polyclonal antibody (Product # PA1-186) at a dilution of 1:2000 overnight at 4C, washed in TBST, and probed with an HRP-conjugated mouse anti-rabbit light chain secondary antibody at a dilution of 1:40,000 for at least 30 minutes. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

NFkB p65 Antibody in ChIP assay (ChIP) — Enrichment of endogenous NF-kappa B p65 protein at specific gene loci using Anti-NF-kappa B p65 Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-NF-kappa B p65 Rabbit Polyclonal Antibody (Product # PA1-186, 2 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.

NFkB p65 Antibody — Modulation of expression of target protein by cell treatment to demonstrate antibody specificity. Western blot analysis of NF-kB p65 using NFkB p65 Polyclonal Antibody (Product # PA1-186) shows enrichment of NF-kB p65 in the nuclear fraction of HeLa cells upon treatment with TNF-alpha, as compared to untreated cells. {TM}

NFkB p65 Antibody in Immunocytochemistry (ICC/IF) — Figure 4 Confocal microscopy for nuclear translocation of p65. (A) T47D cells (B) , MDA-MB-231 cells were pretreated with MyD88 inhibitor for 4 h prior to addition of TLR3 ligand (10 mug/ml) for 60 min. Cells were stained with antibody against p65 subunit of NF-kappaB and Alexa 594-tagged secondary antibody and counterstained with DAPI, and image acquired through confocal microscope (magnification, 63X). (C,D) Bar graph is presented as mean +- S.D for the quantitative measurements of nuclear localization of NF-kappaB at 30, 60, and 90 min of stimulation, analyzed through Image J package ( p < 0.05 is treated as significant). (E,F) Bar graph at 60 min of stimulation showing the highest nuclear localization of NF-kappaB.

NFkB p65 Antibody in Western Blot, Immunocytochemistry (WB, ICC/IF) — Figure 6 Melatonin attenuates NF-kBp65 upregulation by CoCl 2 in microglia. Western blot analysis ( a ) of NF-kBp65 levels in BV2 cells exposed to CoCl 2 (250 muM for 8 h) alone, or in combination with either melatonin (1 muM) or melatonin + EX527 (5 muM). In ( b ), immunostaining of NF-kBp65 (green) and nuclear counterstaining with DAPI (blue) following exposure to CoCl 2 alone (250 muM for 8 h), or in the presence of melatonin (1 muM) + EX527 (5 muM). Bars are mean +- SEM of three independent experiments. * p < 0.05 vs. control (C), deg p < 0.05 vs. CoCl 2 and # p < 0.05 vs. CoCl 2 + MEL by one-way ANOVA followed by Newman-Keuls test for statistical significance. Representative images are shown. Scale bar = 20 mum.

Product Details

Applications

Tested Dilution

Publications

Western Blot (WB)

1-3 µg/mL

View 9 publications 9 publications

Immunocytochemistry (ICC/IF)

1-2 µg/mL

View 2 publications 2 publications

Flow Cytometry (Flow)

3-5 µg/1x10^6 cells

Immunoprecipitation (IP)

3 µg

ChIP assay (ChIP)

1-5 µg x 10^6 cells

Product Specifications

Species Reactivity

Human, Mouse

Published species

Human, Mouse

Host/Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

GLLSGDEDFSSIADMDFS

Conjugate

Unconjugated

Form

Liquid

Concentration

1 mg/mL

Purification

Antigen affinity chromatography

Storage buffer

PBS with 30% glycerol, 1mg/mL BSA

Contains

0.05% sodium azide

Storage conditions

-20°C

RRID

AB_2539917

Product Specific Information

PA1-186 has been successfully used in WB using StartingBlock T20 (TBS) Blocking Buffer (Product # 37543). More non-specific bands are observed when using 5% BSA for blocking.

By WB, PA1-186 detects a predominant band at 65kD. Loading increasing amounts of cell lysate may yield more intense nonspecific bands at ~120kD and/or ~50kD.

PA1-186 has been succesfully used for IP of the NF-kB p65 subunit and co-IP of the NF-kB p50 subunit.

Target Information

Nuclear factor kB p65 (NF-kB p65) is encoded by the RELA gene and is present on chromosome 11 in humans. NF-kB P65 is also known as RelA (v-rel avian reticuloendotheliosis viral oncogene homolog A) and belongs to the Rel family of proteins. It is one of the two subunits of NF-kB (Nuclear factor kappa-light-chain-enhancer of activated B cells) that heterodimerizes with the other subunits p50 or p52. However, binding of TNF alpha to its cognate receptor phosphorylates IKK which in turn phosphorylates I kB allowing proteasomal degradation of I kB. Phosphorylation of p65 at Ser 536 located in the carboxy-terminal transactivation domains (TAD) leads to nuclear localization of the transcriptionally active complex and NF-kB mediated transactivation of several downstream genes. It specifically plays a key role in transcription of immunoglobulin k (kappa) gene in mature B-lymphoid cells.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: avian reticuloendotheliosis viral (v-rel) oncogene homolog A; MGC131774; NF KB; nf-kappa-b p65; NF-kappa-B p65delta3; NF-kappa-B transcription factor p65; NFkappaB p65; nfkb rela; NFkB-p50; NFKB1; nuclear factor; nuclear factor kappa b; Nuclear factor NF-kappa-B p65 subunit; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3; p65 NF kappaB; p65 NF-kappa B; p65 NFkB; REL A; Transcription factor p65; v-rel avian reticuloendotheliosis viral oncogene homolog A; v-rel reticuloendotheliosis viral oncogene homolog A

Gene Aliases: NFKB3; p65; RELA

UniProt ID: (Human) Q04206, (Mouse) Q04207

Entrez Gene ID: (Human) 5970, (Mouse) 19697

Molecular Function: Rel homology transcription factor