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Invitrogen

Phospho-NFkB p65 (Ser529) Monoclonal Antibody (B33B4WP), PerCP-eFluor™ 710, eBioscience™

2 Published Figures
4 References
View all (143) NFkB p65 antibodies

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Product Details

46-9863-42

Applications
Tested Dilution
Publications

Flow Cytometry (Flow)

5 µL (0.03 µg)/test
View 4 publications 4 publications
Product Specifications

Species Reactivity

Human

Published species

Mouse

Host/Isotype

Mouse / IgG2a, kappa

Recommended Isotype Control

Mouse IgG2a kappa Isotype Control (eBM2a), PerCP-eFluor™ 710, eBioscience™

Class

Monoclonal

Type

Antibody

Clone

B33B4WP

Conjugate

PerCP-eFluor™ 710 PerCP-eFluor™ 710 PerCP-eFluor™ 710

Excitation/Emission Max

482/708 nm View spectra spectra

Form

Liquid

Concentration

5 µL/Test

Purification

Affinity chromatography

Storage buffer

PBS, pH 7.2, with BSA

Contains

0.09% sodium azide

Storage conditions

4°C, store in dark, DO NOT FREEZE!

Shipping conditions

Ambient (domestic); Wet ice (international)

RRID

AB_2573916

Product Specific Information

Description: The B33B4WP monoclonal antibody recognizes human NF kappa B (NFkB) p65 subunit when serine 529 is phosphorylated. NFkB, also known as nuclear factor kappa-light chain enhancer of activated B cells, is a ubiquitous transcription factor that regulates the transcription of many genes involved in cell proliferation, apoptosis, development, immunity and cancer. Functional NFkB is a homo- or hetero-dimer composed of 5 members of the NFkB family: p65 (RelA), c-Rel, RelB, p50 (NFkB1, p105 precursor protein), and p52 (NFkB2, p100 precursor protein). The activity of the complex is negatively regulated by binding to IkB inhibitors that sequester NFkB into the cytoplasm, inhibiting its transcriptional activity. NFkB-activating agents like tumor necrosis factor (TNF) alpha, interleukin-1 beta, lipopolysaccharide, camptothecin, and phorbol ester (PMA) induce the phosphorylation and degradation of IkB, leading to the translocation of NFkB to the nucleus where it binds to kB motifs and regulates gene expression. The activity of p65-containing NFkB complexes is positively regulated by phosphorylation of the p65 subunit at serine 529 and/or serine 536.

The B33B4WP monoclonal antibody recognizes a single band at 65 kDa in reduced SDS lysates from TNF alpha-stimulated HeLa cells.

Applications Reported: This B33B4WP antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

Applications Tested: This B33B4WP antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.03 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.

PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.

Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Protocols: We recommend Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins. Alternatively, Protocol C: Two-step protocol: Fixation/Methanol can also be used. Protocol B: One-step protocol: intracellular (nuclear) proteins cannot be used. All Protocols can be found in the "Staining intracellular Antigens for Flow Cytometry Protocol" located in the BestProtocols® Section under the Resources tab online.

Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.

Filtration: 0.2 µm post-manufacturing filtered.

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. 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.

How to use the Panel Builder

Watch the video to learn how to use the Invitrogen Flow Cytometry Panel Builder to build your next flow cytometry panel in 5 easy steps.

Bioinformatics

Protein Aliases: EBP-1; MGC131774; NF KB; nf-kappa-b p65; NF-kappa-B p65delta3; NF-kappa-B transcription factor p65; NF-kB 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; REL A; Transcription factor p65; unnamed protein product; v-rel avian reticuloendotheliosis viral oncogene homolog A

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Gene Aliases: AIF3BL3; CMCU; NFKB3; p65; RELA

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UniProt ID: (Human) Q04206

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Entrez Gene ID: (Human) 5970

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Function(s)
transcription regulatory region sequence-specific DNA binding RNA polymerase II regulatory region sequence-specific DNA binding RNA polymerase II core promoter proximal region sequence-specific DNA binding RNA polymerase II core promoter sequence-specific DNA binding RNA polymerase II transcription factor activity, sequence-specific DNA binding transcription coactivator binding transcriptional repressor activity, RNA polymerase II transcription regulatory region sequence-specific binding transcriptional activator activity, RNA polymerase II transcription regulatory region sequence-specific binding DNA binding chromatin binding double-stranded DNA binding transcription factor activity, sequence-specific DNA binding protein binding enzyme binding protein kinase binding chromatin DNA binding ubiquitin protein ligase binding peptide binding phosphate ion binding identical protein binding protein homodimerization activity actinin binding histone deacetylase binding sequence-specific DNA binding macromolecular complex binding NF-kappaB binding ankyrin repeat binding general transcription initiation factor binding DNA-binding transcription factor binding
Process(es)
negative regulation of transcription from RNA polymerase II promoter negative regulation of cytokine production liver development hair follicle development defense response to tumor cell response to ischemia chromatin organization transcription, DNA-templated regulation of transcription, DNA-templated regulation of transcription from RNA polymerase II promoter defense response inflammatory response cellular defense response neuropeptide signaling pathway I-kappaB kinase/NF-kappaB signaling positive regulation of cell proliferation response to xenobiotic stimulus response to mechanical stimulus response to bacterium organ morphogenesis response to UV-B positive regulation of vascular endothelial growth factor production positive regulation of gene expression positive regulation of Schwann cell differentiation negative regulation of angiogenesis cytokine-mediated signaling pathway signal transduction involved in regulation of gene expression protein catabolic process response to muramyl dipeptide response to lipopolysaccharide response to progesterone positive regulation of interleukin-1 beta production positive regulation of interleukin-12 production positive regulation of interleukin-6 production positive regulation of interleukin-8 production response to insulin tumor necrosis factor-mediated signaling pathway negative regulation of protein sumoylation response to cobalamin response to cytokine toll-like receptor 4 signaling pathway intracellular signal transduction cellular response to hepatocyte growth factor stimulus response to muscle stretch NIK/NF-kappaB signaling vascular endothelial growth factor signaling pathway toll-like receptor TLR6:TLR2 signaling pathway prolactin signaling pathway negative regulation of protein catabolic process response to hydrogen peroxide negative regulation of apoptotic process positive regulation of I-kappaB kinase/NF-kappaB signaling response to amino acid response to morphine innate immune response response to ethanol 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 positive regulation of 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 interleukin-1-mediated signaling pathway response to interleukin-1 cellular response to lipopolysaccharide cellular response to lipoteichoic acid cellular response to peptidoglycan cellular response to nicotine cellular response to interleukin-1 cellular response to interleukin-6 cellular response to tumor necrosis factor postsynapse to nucleus signaling pathway antiviral innate immune response negative regulation of cytokine production involved in inflammatory response negative regulation of NIK/NF-kappaB signaling positive regulation of NIK/NF-kappaB signaling positive regulation of beta-amyloid formation negative regulation of pri-miRNA transcription from RNA polymerase II promoter positive regulation of pri-miRNA transcription from RNA polymerase II promoter cellular response to angiotensin positive regulation of leukocyte adhesion to vascular endothelial cell positive regulation of miRNA metabolic process negative regulation of extrinsic apoptotic signaling pathway
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