Invitrogen

NFkB p65 Polyclonal Antibody

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This Antibody was verified by IP Mass Spectrometry to ensure that the antibody binds to the antigen stated. View Details

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NFkB p65 Antibody in Cell Treatment — 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 IP-MS — IP-MS enrichment of RELA (LFQ intensity): RELA was enriched 444-fold from A549 lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Product # 90409) and RELA antibody (Product # PA1-186). The STRING database (www.string-db.org) was used to identify the protein interactor list. See more information on IP-MS verification of antibody selectivity. {IP-MS}

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 in Immunoprecipitation (IP) — NF-kB p65 was immunoprecipitated using 3 µg of the NF-kB p65 Rabbit Polyclonal Antibody (Product # PA1-186) from lysate of HeLa (Lane 3) using the Dynabeads® Protein A Immunoprecipitation Kit (Product # 10006D). Normal Rabbit IgG was used as a Isotype control (Lane 2). 10 % input represents the cell extract used for immunoprecipitation (Lane 1). Western blot analysis was performed using NF-kB p65 Rabbit Polyclonal Antibody (Product # PA1-186) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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) — Co-Immunoprecipitation of NF-kB p50 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 p50 rabbit monoclonal antibody at a dilution of 1:1000 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 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 Western Blot (WB) — Western blot analysis of NF-kB p65 was performed by loading 20 µg of cytoplasmic (cyto) and nuclear extracts from HeLa cells either left untreated (UT) or treated with 25 ng/mL of recombinant TNF-alpha (Product # RTNFAI) for 35 minutes at 37C, and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were 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. NF-kB p65 was detected at 65 kD using an NF-kB p65 polyclonal antibody (Product # PA1-186) at a dilution of 1:2000 in StartingBlock T20 (TBS) Blocking Buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-rabbit IgG secondary antibody (Product # 31460) at a dilution of 1:40,000 for 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

NFkB p65 Antibody in Western Blot (WB) — Western blot analysis of NF-kB p65 was performed by loading 25 µg of the indicated whole cell lysates, and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were 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. NF-kB p65 was detected at 65 kD using an NF-kB p65 polyclonal antibody (Product # PA1-186) at a dilution of 1:2000 in StartingBlock T20 (TBS) Blocking Buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated mouse anti-rabbit light-chain secondary antibody at a dilution of 1:40,000 for 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

NFkB p65 Antibody — Fig. 5. FSTL1 overexpression rescues the miR-27a-mediated suppressive effect on RA-FLS migration and invasion by activating the TLR4/NFkappaB pathway. RA-FLS were transfected with miR-27a, exogenous FSTL1, or miR-27a and exogenous FSTL1. (A) The cell migration and invasion of RA-FLS as measured using the Transwell system. (B) Protein expression levels of TLR4 and NFkappaB in RA-FLS were detected by western blot analysis. * p < 0.05, versus control mimic group. # p < 0.05, versus Ad-beta-gal group. a p < 0.05, versus miR-27a mimic group.

Product Details

Tested Applications

Dilution

ChIP assay (ChIP)

1-5 µg x 10^6 cells

Flow Cytometry (Flow)

3-5 µg/1x10^6 cells

Immunocytochemistry (ICC)

1-2 µg/mL

Immunofluorescence (IF)

1-2 µg/mL

Immunoprecipitation (IP)

3 µg

Western Blot (WB)

1-3 µg/mL

Published Applications

Western Blot (WB)

See 3 publications below

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

NF-kB (nuclear factor kB) regulates the expression of a large number of genes that play critical roles in apoptosis, viral replication, tumorigenesis, various autoimmune diseases, and inflammation. The active nuclear form of the NF-kB transcription factor complex is composed of two DNA binding subunits, NF-kB p65 and p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65.

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; NF-kappa-B p65delta3; NF-kappa-B transcription factor p65; NFKB3; Nuclear Factor kB p65 subunit; nuclear factor NF-kappa-B p65 subunit; nuclear factor of kappa light polypeptide gene enhancer in B-cells 3; p65; p65 NF kappaB; p65 NF-kappa B; p65 NFkB; RelA; Transcription Factor p65; v-rel avian reticuloendotheliosis viral oncogene homolog A; v-rel reticuloendotheliosis viral oncogene homolog A

Gene Aliases: NFKB3; p65

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

Molecular Function: nucleic acid binding transcription factor

negative regulation of transcription from RNA polymerase II promoter liver development hair follicle development stimulatory C-type lectin receptor signaling pathway acetaldehyde metabolic process transcription from RNA polymerase II promoter inflammatory response cellular defense response aging positive regulation of cell proliferation organ morphogenesis response to organic substance response to UV-B positive regulation of Schwann cell differentiation cytokine-mediated signaling pathway membrane protein intracellular domain proteolysis positive regulation of chondrocyte differentiation positive regulation of type I interferon production response to muramyl dipeptide response to progesterone response to insulin response to cobalamin cellular response to hepatocyte growth factor stimulus response to muscle stretch Fc-epsilon receptor signaling pathway negative regulation of protein catabolic process response to drug negative regulation of apoptotic process positive regulation of I-kappaB kinase/NF-kappaB signaling response to amino acid response to morphine positive regulation of interleukin-12 biosynthetic process negative regulation of transcription, DNA-templated positive regulation of transcription, DNA-templated positive regulation of transcription from RNA polymerase II promoter negative regulation of insulin receptor signaling pathway regulation of inflammatory response T cell receptor signaling pathway positive regulation of NF-kappaB transcription factor activity response to cAMP defense response to virus cellular response to hydrogen peroxide nucleotide-binding oligomerization domain containing 2 signaling pathway response to interleukin-1 cellular response to lipopolysaccharide cellular response to nicotine cellular response to interleukin-1 cellular response to interleukin-6 cellular response to tumor necrosis factor cellular response to peptide hormone stimulus negative regulation of NIK/NF-kappaB signaling positive regulation of miRNA metabolic process negative regulation of extrinsic apoptotic signaling pathway transcription, DNA-templated regulation of transcription, DNA-templated regulation of transcription from RNA polymerase II promoter defense response I-kappaB kinase/NF-kappaB signaling response to bacterium response to cytokine NIK/NF-kappaB signaling innate immune response

Process(es)

RNA polymerase II regulatory region sequence-specific DNA binding RNA polymerase II core promoter proximal region sequence-specific DNA binding RNA polymerase II distal enhancer sequence-specific DNA binding transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding transcriptional repressor activity, RNA polymerase II core promoter proximal region sequence-specific binding transcriptional activator activity, RNA polymerase II distal enhancer sequence-specific binding DNA binding chromatin binding transcription factor activity, sequence-specific DNA binding transcription factor activity, RNA polymerase II distal enhancer sequence-specific binding protein binding transcription factor binding protein kinase binding chromatin DNA binding ubiquitin protein ligase binding protein complex binding activating transcription factor binding phosphate ion binding identical protein binding protein homodimerization activity actinin binding histone deacetylase binding transcription regulatory region DNA binding protein heterodimerization activity protein N-terminus binding NF-kappaB binding repressing transcription factor binding ankyrin repeat binding enzyme binding sequence-specific DNA binding

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