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AB324465

NFkB p65 Transcription Factor Activity Assay Kit

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ab324465 NFkB p65 Transcripiton Factor Activity Assay is s single-wash 90 minute DNA Sandwich ELISA designed for the semi-quantiatve measurement of activated NFkB p65 in nuclear extracts, cell lysates and tissue lysates.

View Alternative Names

NFKB3, RELA, Transcription factor p65, Nuclear factor NF-kappa-B p65 subunit, Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3

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Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)
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Supplier Data

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)

A competition assay using the Competitor DNA Probe containing the wild-type consensus sequence and Non-Competitor DNA Probe containing a mutated consensus sequence are used to demonstrate assay specificity. The Competitor DNA Probe will inhibit NFkB p65 binding to the Capture DNA Probe immobilized on the plate, while the Non-Competitor DNA Probe should have little to no effect on NFkB binding. 3 µg/well Nuclear Extracts from Hela cells treated with 25 ng/mL TNF alpha was assayed in each condition. 20 pmol Competitor DNA Probe was added to competitor wells. 20 pmol Non-competitor DNA Probe was added to non-competitor wells.

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)
  • FuncS

Supplier Data

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)

Abcam’s Transcription Factor Activity Assays is a single wash 90-minute DNA sandwich ELISA. Sample and HRP conjugated antibody are applied to the oligonucleotide coated plate in a single reaction mix. After 1 hour, wash and add TMB substrate for 15 minutes. Add stop solution and read at 450 nm.

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)
  • FuncS

Supplier Data

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)

Nuclear extracts from Hela cells and Hela cells treated with 25 ng/mL TNF alpha were assayed from 0.19 - 24 µg/well using the Abcam NFkB p65 Transcription Factor Activity Assay. Data shown is from wells assayed in duplicate. Comparison of treated to untreated Hela nuclear extract shows average fold change ~27. ab219177 Nuclear Extraction kit was used to extract nuclear proteins.

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)
  • FuncS

Supplier Data

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)

TNF alpha treated Hela nuclear extract was assayed from 0.38 – 12 µg with two NFkB p65 transcription factor kits. ab324465 90-minute assay is more sensitive when compared to ab133112 conventional 3.5-hour assay. It requires less time and sample but delivers robust data.

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)
  • FuncS

Supplier Data

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)

Whole cell lysate from Jurkat cells and Jurkat cells treated with PMA and Ionomycin were assayed from 2.5 - 20 µg/well using the Abcam NFkB p65 Transcription Factor Activity Assay. Data shown is from wells assayed in duplicate. Whole cell lysate was extracted following assay protocol.

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)
  • FuncS

Supplier Data

Functional Studies - NFkB p65 Transcription Factor Activity Assay Kit (AB324465)

Lysate from Human Colon tissue was spiked with TNF alpha treated Hela Nuclear extract. Spiked Hu colon lysate was assayed from 0.19 – 12 µg/well using the Abcam NFkB p65 Transcription Factor Activity Assay. Data show is from wells assayed in duplicate. Tissue was extracted following assay protocol. Spiking into human colon lysate demonstrates assay compatibility with human tissues samples.

Key facts

Detection method

Colorimetric

Sample types

Nuclear Extracts, Cell Extract, Tissue Lysate

Assay type

Semi-quantitative

Sensitivity

> 0.1875 µg

Assay time

1h 30m

Assay Platform

Pre-coated microplate (12 x 8 well strips)

Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Reactivity", "Dilution Info", "Notes"] }, "values": { "FuncS": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }

Product details

ab324465 NFkB p65 Transcripiton Factor Activity Assay is s single-wash 90 minute DNA Sandwich ELISA designed for the semi-quantiatve measurement of activated NFkB p65 in nuclear extracts, cell lysates and tissue lysates.

A combination of proprietary technology to streamline a typically 4 hour protocol to a single wash 90 minute format. A double stranded DNA probe containing the NFkB connsensus binding site is coated on the 96-well plate. Active NFkB p65 in the sample binds to the DNA probe on the plate. After the addition of an HRP conjugated detector antibody that recognizes NFkB p65 bound to the probe, TMB substrate is added and a color signal is developed, which is measured at 450 nm. A reaction mix containing binding buffer, sample and HRP conjugated antibody is added to plate and incubated for 1 hour. After which the plate is washed and TMB applied. TMB develops for 5-20 mins, then stop is applied. Read at 450 nm.

Precision

[ { "reproducibilityType": "Intra", "sample": "Nuclear Extracts", "replicates": 8, "mean": null, "standardDeviation": null, "coefficientOfVariability": "= 2" }, { "reproducibilityType": "Inter", "sample": "Nuclear Extracts", "replicates": 3, "mean": null, "standardDeviation": null, "coefficientOfVariability": "= 9" } ]

What's included?

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Properties and storage information

Shipped at conditions
Blue Ice
Appropriate short-term storage conditions
-20°C
Appropriate long-term storage conditions
-20°C
Storage information
-20°C

Product protocols

Target data

NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52. The heterodimeric RELA-NFKB1 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. The NF-kappa-B heterodimeric RELA-NFKB1 and RELA-REL complexes, for instance, function as transcriptional activators. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. The inhibitory effect of I-kappa-B on NF-kappa-B through retention in the cytoplasm is exerted primarily through the interaction with RELA. RELA shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Beside its activity as a direct transcriptional activator, it is also able to modulate promoters accessibility to transcription factors and thereby indirectly regulate gene expression. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1. Essential for cytokine gene expression in T-cells (PubMed : 15790681). The NF-kappa-B homodimeric RELA-RELA complex appears to be involved in invasin-mediated activation of IL-8 expression. Key transcription factor regulating the IFN response during SARS-CoV-2 infection (PubMed : 33440148).
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