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AB324202

NFkB p50 Transcription Factor Activity Assay Kit

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ab324202 NFkB p50 Transcription Factor Activity Assay Kit is a single-wash 90 minute DNA Sandwich ELISA designed for the semi-quantitative measurement of activated NFkB p50 in nuclear extracts, cell lysates and tissue lysates.

View Alternative Names

Nuclear factor NF-kappa-B p105 subunit, DNA-binding factor KBF1, EBP-1, Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, NFKB1

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Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)
  • sELISA

Supplier Data

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)

Transcription Factor Activity Assay Flow.

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.

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)
  • sELISA

Supplier Data

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)

NFkB p50 activity in Human lung lysate.

Lysate from Human lung tissue was assayed from 2.5 µg-50 µg/well using the Abcam NFkB p50 Transcription Factor Activity Assay. Data show is from wells assayed in duplicate. Tissue was extracted following assay protocol.

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)
  • sELISA

Supplier Data

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)

Specificity of NFkB p50 Transcription Factor Activity Assay.

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 p50 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.

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)
  • sELISA

Supplier Data

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)

NFkB p50 activity in treated and untreated Jurkat whole cell lysate.

Whole cell lysate from Jurkat cells and Jurkat cells treated with PMA and Ionomycin were assayed from 2.5 µg-10 µg/well using the Abcam NFkB p50 Transcription Factor Activity Assay. Data shown is from wells assayed in duplicate. Whole cell lysate was extracted following assay protocol.

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)
  • sELISA

Supplier Data

Sandwich ELISA - NFkB p50 Transcription Factor Activity Assay Kit (AB324202)

NFkB p50 activity in treated and untreated Hela nuclear extract.

Nuclear extracts from Hela cells and Hela cells treated with 25 ng/mL TNF alpha were assayed from 0.1875 µg-12 µg/well using the Abcam NFkB p50 Transcription Factor Activity Assay. Data shown is from wells assayed in duplicate. Comparison of treated to untreated Hela nuclear extract shows average fold change ~17. ab219177 Nuclear Extraction kit was used to extract nuclear proteins.

Key facts

Detection method

Colorimetric

Sample types

Nuclear Extracts, Cell Extract, Tissue Lysate

Reacts with

Human

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": { "sELISA": { "reactivity":"TESTED_AND_REACTS", "dilution-info":"", "notes":"<p></p>" } } }

Product details

ab324202 NFkB p50 Transcription Factor Activity Assay Kit is a single-wash 90 minute DNA Sandwich ELISA designed for the semi-quantitative measurement of activated NFkB p50 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 consensus binding site is coated on the 96-well plate. Active NFkB p50 in the sample binds to the DNA probe on the plate. After the addition of an HRP conjugated detector antibody that recognizes NFkB p50 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 solution 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 and the heterodimeric p65-p50 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. 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. NF-kappa-B heterodimeric p65-p50 and RelB-p50 complexes are transcriptional activators. The NF-kappa-B p50-p50 homodimer is a transcriptional repressor, but can act as a transcriptional activator when associated with BCL3. NFKB1 appears to have dual functions such as cytoplasmic retention of attached NF-kappa-B proteins by p105 and generation of p50 by a cotranslational processing. The proteasome-mediated process ensures the production of both p50 and p105 and preserves their independent function, although processing of NFKB1/p105 also appears to occur post-translationally. p50 binds to the kappa-B consensus sequence 5'-GGRNNYYCC-3', located in the enhancer region of genes involved in immune response and acute phase reactions. In a complex with MAP3K8, NFKB1/p105 represses MAP3K8-induced MAPK signaling; active MAP3K8 is released by proteasome-dependent degradation of NFKB1/p105.. Nuclear factor NF-kappa-B p105 subunit. P105 is the precursor of the active p50 subunit (Nuclear factor NF-kappa-B p50 subunit) of the nuclear factor NF-kappa-B (PubMed : 1423592). Acts as a cytoplasmic retention of attached NF-kappa-B proteins by p105 (PubMed : 1423592).. Nuclear factor NF-kappa-B p50 subunit. Constitutes the active form, which associates with RELA/p65 to form the NF-kappa-B p65-p50 complex to form a transcription factor (PubMed : 1740106, PubMed : 7830764). Together with RELA/p65, binds to the kappa-B consensus sequence 5'-GGRNNYYCC-3', located in the enhancer region of genes involved in immune response and acute phase reactions (PubMed : 1740106, PubMed : 7830764).
See full target information NFKB1
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