NFkB p65 Transcription Factor Assay Kit ab133112 is a non-radioactive, sensitive ELISA-based method for detecting specific transcription factor NF-kB (p65) DNA binding activity in nuclear extracts.
- No cross-reactivity with NF-kB (p50)
- Colorimetric assay - 450nm readout; works on any plate reader
- Cited in over 95 publications
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- Environmental toxicology 39:3253-32632024Ellagic acid protects against angiotensin II-induced hypertrophic responses through ROS-mediated MAPK pathway in H9c2 cells.Applications:Unspecified applicationReactive species:Unspecified reactive speciesYa-Che Lee,Yeong-Chin Jou,Wan-Ching Chou,Kun-Ling Tsai,Cheng-Huang Shen,Shin-Da LeePubMed 38356441
- Cell death & disease 14:7432023BRISC is required for optimal activation of NF-κB in Kupffer cells induced by LPS and contributes to acute liver injury.Applications:Unspecified applicationReactive species:Unspecified reactive speciesWen Zhang,Kai Liu,Guang-Ming Ren,Yu Wang,Ting Wang,Xian Liu,Dong-Xu Li,Yang Xiao,Xu Chen,Ya-Ting Li,Yi-Qun Zhan,Shen-Si Xiang,Hui Chen,Hui-Ying Gao,Ke Zhao,Miao Yu,Chang-Hui Ge,Chang-Yan Li,Zhi-Qiang Ge,Xiao-Ming Yang,Rong-Hua YinPubMed 37968261
- iScience 26:107997202315-keto-PGE alleviates nonalcoholic steatohepatitis through its covalent modification of NF-κB factors.Applications:Unspecified applicationReactive species:Unspecified reactive speciesSiow-Wey Hee,Yi-Cheng Chang,Lynn Su,Ing-Jung Chen,Yung-Ming Jeng,Meng-Lun Hsieh,Yu-Chia Chang,Fu-An Li,Daniel Liao,Shiau-Mei Chen,Lee-Ming ChuangPubMed 37810249
- Environmental toxicology 38:2165-21722023Sesamin suppresses angiotensin-II-enhanced oxidative stress and hypertrophic markers in H9c2 cells.Applications:Unspecified applicationReactive species:Unspecified reactive speciesChih-Chia Chang,Hui-Ching Cheng,Wan-Ching Chou,Yu-Ting Huang,Pei-Ling Hsieh,Pei-Ming Chu,Shin-Da LeePubMed 37357850
- Oncology reports 50:2023The MyD88 inhibitor, ST2825, induces cell cycle arrest and apoptosis by suppressing the activation of the NF‑κB/AKT1/p21 pathway in pancreatic cancer.Applications:Unspecified applicationReactive species:Unspecified reactive speciesSinan Lu,Tianyu He,Yuan Zhang,Bo Zhou,Qiyi Zhang,Sheng YanPubMed 37326109
- Natural product communications 18:2023Augmentation of Docetaxel-Induced Cytotoxicity in Human PC-3 Androgen-Independent Prostate Cancer Cells by Combination With Four Natural Apoptosis-Inducing Anticancer Compounds.Applications:Unspecified applicationReactive species:Unspecified reactive speciesInass A Ahmed,Saly Hafiz,Sabrina van Ginkel,Satyanarayana R Pondugula,Azza S Abdelhaffez,Hayam G Sayyed,Ebtihal A Abd El-Aziz,Mahmoud M MansourPubMed 37292146
- Frontiers in nutrition 9:10786422023Targeting a transcription factor NF-κB by green tea catechins using and studies in pancreatic cancer.Applications:Unspecified applicationReactive species:Unspecified reactive speciesMohd Suhail,Mohd Rehan,Mohammad Tarique,Shams Tabrez,Amjad Husain,Torki A ZughaibiPubMed 36712528
- PLoS genetics 18:e10105062022Aberrant expression and localization of the RAP1 shelterin protein contribute to age-related phenotypes.Applications:Unspecified applicationReactive species:Unspecified reactive speciesAmanda J Stock,Ross A McDevitt,Chandrakala Puligilla,Yajun Wang,Yongqing Zhang,Kun Wang,Chongkui Sun,Kevin G Becker,Elin Lehrmann,William H Wood,Yi Gong,Mohammad Aqdas,Myong-Hee Sung,Victoria Hoffmann,Chengyu Liu,Myriam Gorospe,Lea Harrington,Luigi Ferrucci,Yie LiuPubMed 36441670
- Current issues in molecular biology 44:5390-54042022Cardamom Extract Alleviates the Oxidative Stress, Inflammation and Apoptosis Induced during Acetaminophen-Induced Hepatic Toxicity via Modulating Nrf2/HO-1/NQO-1 Pathway.Applications:Unspecified applicationReactive species:Unspecified reactive speciesEssraa A R Alkhalifah,Amjad A Alobaid,Marwah A Almajed,Manar K Alomair,Lama S Alabduladheem,Sarah F Al-Subaie,Abdullah Akbar,Mahesh V Attimarad,Nancy S Younis,Maged E MohamedPubMed 36354677
- Pharmaceutics 14:2022The Effects of Resveratrol-Rich Extracts of Pruning Waste on HeLa, MCF-7 and MRC-5 Cells: Apoptosis, Autophagia and Necrosis Interplay.Applications:Unspecified applicationReactive species:Unspecified reactive speciesAleksandra Jovanović Galović,Nataša Jovanović Lješković,Senka Vidović,Jelena Vladić,Nikola Jojić,Milan Ilić,Tatjana Srdić Rajić,Vesna Kojić,Dimitar JakimovPubMed 36297452
Key facts
- Detection method
- Colorimetric
- Sample types
- Nuclear Extracts
- Assay type
- Semi-quantitative
- Reactive species
- Mouse, Rat, Human
- Assay time
- 3h 30m
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Target data
Function
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).
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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NFkB p65 Transcription Factor Assay Kit ab133112 is a non-radioactive, sensitive ELISA-based method for detecting specific transcription factor NF-kB (p65) DNA binding activity in nuclear extracts.
- No cross-reactivity with NF-kB (p50)
- Colorimetric assay - 450nm readout; works on any plate reader
- Cited in over 95 publications
Key facts
- Detection method
- Colorimetric
- Sample types
- Nuclear Extracts
- Assay type
- Semi-quantitative
- Reactive species
- Mouse, Rat, Human
- Assay time
- 3h 30m
- Assay Platform
- Microplate reader
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- Multi
- Appropriate long-term storage conditions
- Multi
- Storage information
- Please refer to protocols
Notes
NFkB p65 Transcription Factor Assay Kit ab133112 is a non-radioactive, sensitive ELISA-based method for detecting specific transcription factor DNA binding activity in nuclear extracts.
In the NFkB p65 assay, a double stranded DNA sequence containing the NFkB response element is immobilized onto the bottom of the wells of a 96-well plate. NFkB contained in a nuclear extract, binds to the NFkB response element, and is detected using an anti NFkB p65 antibody. A secondary antibody conjugated to HRP is added to provide a colorimetric readout at 450 nm.
NFkB p65 transcription factor assay protocol summary:
- prepare nuclear extracts from cells
- add samples to wells
- incubate for 1 hr or o/n
- wash with wash buffer
- add NFkB antibody and incubate for 1 hr , then wash
- add HRP-conjugated secondary antibody and incubate for 1 hr, then wash
- add developing solution and incubate for 15-45 min
- add stop solution
- analyze with microplate reader
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
NF-kB p65 also known as RelA is a significant component of the NF-kB protein complex. This complex usually involves a molecular weight for p65 of approximately 65 kDa. NF-kB p65 is mechanistically a transcription factor that regulates genes involved in inflammation cell survival and immune response. Expression of p65 is widely seen in various cell types including immune cells and epithelial cells suggesting its role in numerous physiological processes.
- Biological function summary
NF-kB p65 acts as part of the larger NF-kB complex usually forming a heterodimer with other family members like p50. This complex translocates to the nucleus upon activation where it binds specific DNA sequences to regulate gene expression. The p65 subunit is essential for transactivating target genes involved in immune and inflammatory responses. Its regulation is important for proper cellular functioning especially in the maintenance of immune homeostasis and inflammatory response.
- Pathways
NF-kB p65 participates in critical pathways like the NF-kB signaling and Toll-like receptor pathways. These pathways are fundamental for initiating immune responses and contribute to the regulation of apoptosis and cellular stress responses. In the context of these pathways the IKK complex plays a pivotal role in NF-kB activation leading to the phosphorylation and subsequent degradation of inhibitors that retain NF-kB p65 in the cytoplasm therefore allowing its movement to the nucleus.
- Associated diseases and disorders
NF-kB p65 is implicated in conditions such as cancer and autoimmune diseases. Its dysregulation can lead to persistent activation of inflammatory pathways contributing to tumorigenesis and chronic inflammation. In the context of these diseases proteins such as the p50 subunit and the IKK complex are often involved reflecting the importance of tight regulation of NF-kB signaling in preventing pathological conditions.
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4 product images
Saha D et al., PLoS One, 12(2). Fig 9a. doi: 10.1371/journal.pone.0171084 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/Functional Studies - NFkB p65 Transcription Factor Assay Kit (ab133112)
After the treatment with LPS (10 μg/ml for 6 hrs), cells were lysed with hypotonic HEPES lysis buffer (pH 7.4) and centrifuged at 1000 g for 10 min at 4°C, supernatants were collected and used for the determination of intracellular p65- NF-κB by ELISA. The absorbance was read at 450 nm using spectrophotometer.
Functional Studies - NFkB p65 Transcription Factor Assay Kit (ab133112)
Jurkat cells were treated with PMA and ionomycin (+). Nuclear lysates were extracted (Nuclear Extraction Kit ab113474) and 40 uL, corresponding to 4e6 cells, were tested in duplicates (+/- SD).
Functional Studies - NFkB p65 Transcription Factor Assay Kit (ab133112)
Titration of positive control with or without inhibitor, background signal subtracted (duplicates; +/- SD).
Functional Studies - NFkB p65 Transcription Factor Assay Kit (ab133112)
Assay of cell lysates isolated from stimulated (20 ng/ml TNF alpha for 30 minutes) and nonstimulated HeLa cells demonstrating NFkB (p65) activity.
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