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Nf κb p65 transcription factor assay kit

Manufactured by Abcam
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Sourced in United Kingdom, United States

The NF-κB p65 Transcription Factor Assay Kit is a laboratory tool designed to detect and quantify the activation of the NF-κB p65 transcription factor. The kit provides a simple and sensitive method for measuring NF-κB p65 DNA-binding activity in nuclear extracts.

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Lab products found in correlation

Nuclear extraction kit, by Abcam (10 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (8 mentions) Synergy ht, by Agilent Technologies (3 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Enspire, by PerkinElmer (1 mentions) Enzyme linked immunosorbent assay kit, by R&D Systems (1 mentions) Dimension clinical chemistry system, by Siemens (1 mentions) N per nuclear and cytoplasmic extraction reagents, by Thermo Fisher Scientific (1 mentions) Epigallocatechin gallate (egcg), by Merck Group (1 mentions) Celltiter 96 aqueous one solution kit, by Promega (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) Elisa kit, by MBL Life Science (1 mentions) Elisa kit, by R&D Systems (1 mentions) Infinite m200 pro, by Tecan (1 mentions) Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Microplate reader, by Bio-Rad (1 mentions) Victor3, by PerkinElmer (1 mentions) Tnf α, by R&D Systems (1 mentions) Microplate reader, by Tecan (1 mentions) Ifn γ, by R&D Systems (1 mentions)

57 protocols using nf κb p65 transcription factor assay kit

1

NF-κB Activation Dynamics in ZNRF4 Knockdown

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HCT116 cells were treated with ZNRF4 siRNA or NT siRNA for 48 h followed by MDP stimulation for various time points. At each time point, cells were collected, nuclear extract was prepared and the DNA-binding activity of NF-κB/p65 was assessed using NF-κB p65 Transcription Factor Assay Kit (Abcam; ab133112) according to the manufacturer’s instructions.
Bist P., Cheong W.S., Ng A., Dikshit N., Kim B.H., Pulloor N.K., Khameneh H.J., Hedl M., Shenoy A.R., Balamuralidhar V., Malik N.B., Hong M., Neutzner A., Chin K.C., Kobayashi K.S., Bertoletti A., Mortellaro A., Abraham C., MacMicking J.D., Xavier R.J, & Sukumaran B. (2017). E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP. Nature Communications, 8, 15865.
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2

Quantifying NF-κB Activity in Colon Tissues

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Extraction of nuclear protein from different colon tissues which treated by DSS or AOM/DSS. Then exam the NF-κB activity using the NF-κB p65 Transcription Factor Assay Kit(ab133112 abcam) following the manufacturer’s instruction. Read absorbance at 450 nm by Microplate Reader (EnSpire, PerkinElmer, USA).
Liu C., Liu E.D., Meng Y.X., Dong X.M., Bi Y.L., Wu H.W., Jin Y.C., Zhao K., Li J.J., Yu M., Zhan Y.Q., Chen H., Ge C.H., Yang X.M, & Li C.Y. (2017). Keratin 8 reduces colonic permeability and maintains gut microbiota homeostasis, protecting against colitis and colitis-associated tumorigenesis. Oncotarget, 8(57), 96774-96790.
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3

NF-κB Activity Assay in Cardiac Fibroblasts

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NF‐κB activity was measured by NF‐κB p65 Transcription Factor Assay Kit (Abcam) according to the manufacturer's protocol. Briefly, nuclear extracts were prepared from cardiac fibroblasts and applied to the assay plate coated by dsDNA with p65 binding region at 4℃ overnight. After washing, the plate was incubated with primary antibody (rabbit anti‐p65, 1:100; attachment of the kit) for 2 hours at room temperature, followed by the incubation with HRP conjugated secondary antibody (goat anti‐rabbit IgG, 1:100; attachment of the kit) for 2 hours at room temperature. After developing reaction for 20 minutes, the light absorbance (450 nm) was detected with EMax Plus.
Tanaka S., Imaeda A., Matsumoto K., Maeda M., Obana M, & Fujio Y. (2020). β2‐adrenergic stimulation induces interleukin‐6 by increasing Arid5a, a stabilizer of mRNA, through cAMP/PKA/CREB pathway in cardiac fibroblasts. Pharmacology Research & Perspectives, 8(2), e00590.
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4

NF-κB DNA Binding Activity Assay

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Nuclear fractions of treated monocytes were extracted, and the DNA binding activity of NF-κB was detected using NF-κB p65 Transcription Factor Assay Kit (ab133112, Abcam, Cambridge, MA, USA) in accordance with manufacturer’s instructions.
Xu H., Shi Q., Mo Y., Wu L., Gu J, & Xu Y. (2019). Downregulation of α7 nicotinic acetylcholine receptors in peripheral blood monocytes is associated with enhanced inflammation in preeclampsia. BMC Pregnancy and Childbirth, 19, 188.
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5

Nuclear NF-κB activation in CAF cells

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CAF cells 10–32 PC Puro were plated at a density of 2×106 on a 100 mm dish in complete media. The following day cells were washed with HBSS and treated with HPNE and HPNE-KRAS cell condition media, or serum-free media, media containing 10 ng/mL CXCL8 and 10 ng/mL CXCL8 with 50 μg/mL of CXCR2 SCH-527123 for two hours. Cells were lysed to obtain nuclear fraction as described in the nuclear extraction kit (NBP2–29447, Novus Biologicals, CO, USA). A part of the obtained nuclear fraction protein was quantified using the BCA kit (Pierce™ BCA Protein Assay Kit). Furthermore, the nuclear fraction was utilized to perform NFκB p65 transcription factor assay as per the protocol given by the NFκB p65 transcription factor assay kit (ab133112, Abcam, MA, USA). The results were normalized to the amount of protein quantified.
Awaji M., Saxena S., Wu L., Prajapati D.R., Purohit A., Varney M.L., Kumar S., Rachagani S., Ly Q.P., Jain M., Batra S.K, & Singh R.K. (2020). CXCR2 signaling promotes secretory cancer-associated fibroblasts in pancreatic ductal adenocarcinoma. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(7), 9405-9418.
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6

Assessing Inflammatory Pathways in Tissue Samples

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Well-known cytokines such as NF-κB and CXCL1, which were associated with HDAC2 and neutrophilic inflammation, were measured in this study. Nuclear proteins were extracted and collected from tissue samples using a nuclear extraction kit (Abcam, Cambridge, UK). The activity of NF-κB was assessed using a NF-κB p65 transcription factor assay kit (Abcam) following the manufacturer’s protocol. We measured the levels of pro-inflammatory cytokines in the BAL fluid, such as CXCL1. Enzyme-linked immunosorbent assay kits were used for the analyses (R&D Systems, Minneapolis, MN, USA).
An T.J., Kim J.H., Hur J., Park C.K., Lim J.U., Kim S., Rhee C.K, & Yoon H.K. (2023). Tiotropium Bromide Improves Neutrophilic Asthma by Recovering Histone Deacetylase 2 Activity. Journal of Korean Medical Science, 38(12), e91.
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7

hs-CRP and NFκB Transcription Assay

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High-sensitive C-reactive protein (hs-CRP) was measured by immunoturbidimetry in an automatic analyzer (SIEMENS Dimension Clinical Chemistry System). Nuclear factor enhancer of the kappa light chains of activated B cells (NFκB) transcription factor was measured by a sandwich ELISA with the NFκB p65 Transcription Factor Assay Kit (ab176648; abcam, USA), which quantifies the p65 fraction in nuclear extracts of mononuclear cells.
Galvis-Pérez Y., Marín-Echeverri C., Franco Escobar C.P., Aristizábal J.C., Fernández M.L, & Barona-Acevedo J. (2020). Comparative Evaluation of the Effects of Consumption of Colombian Agraz (Vaccinium meridionale Swartz) on Insulin Resistance, Antioxidant Capacity, and Markers of Oxidation and Inflammation, Between Men and Women with Metabolic Syndrome. BioResearch Open Access, 9(1), 247-254.
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8

NF-κB p65 DNA Binding Assay

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The NF-κB p65-DNA binding was assessed using a NF-κB p65 Transcription Factor Assay Kit (ab133112, Abcam, UK). The nuclear lysate was used to quantify the relative nuclear NF-κB p65-DNA binding. Briefly, a specific double stranded DNA sequence containing the NF-κB responsive binding element was immobilized onto the bottom of wells of a 96-well plate. p65 in the nuclear extract recognized and bound to the NF-κB responsive binding element. p65 was detected using a specific primary antibody against p65, and the signal was enhanced using a secondary antibody conjugated with horseradish peroxidase (HRP). The absorbance was read at 450 nm. NF-κB p65 non-specific competitor dsDNA served as positive and negative controls, respectively. Each experiment was repeated at least three times.
Lee J.H., Jo E.H., Lee B., Noh H.M., Park S., Lee Y.M., Kim D.K, & Park M.C. (2019). Soshiho-Tang, a Traditional Herbal Medicine, Alleviates Atopic Dermatitis Symptoms via Regulation of Inflammatory Mediators. Frontiers in Pharmacology, 10, 742.
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9

Quantifying NF-κB Activation in CTPE Cells

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To measure NFκB activation in control and Pso-treated CTPE cells, nuclear extracts were prepared using N-PER-Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer’s protocol. The NFκB p65 Transcription Factor Assay Kit (Abcam, Cambridge, MA, USA) was used to detect NFκB activation according to the manufacturer’s protocol.
Pal D., Suman S., Kolluru V., Sears S., Das T.P., Alatassi H., Ankem M.K., Freedman J.H, & Damodaran C. (2017). Inhibition of autophagy prevents cadmium-induced prostate carcinogenesis. British Journal of Cancer, 117(1), 56-64.
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10

Quantifying Nuclear NF-κB Activation

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After incubation, the nuclei and cytosol of cultured Sw.71 were separated using a nuclear extraction kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Generally, inactive NF-κB complexes are existed in the cytoplasm, whereas active NF-κB complexes (p65) translocate to the nucleus [10 (link)]. Therefore, the expression of NF-κB p65 levels in isolated nuclei and cytosol were analyzed using the NF-κB p65 transcription factor assay kit (Abcam) according to the manufacturer’s instructions [35 (link)]. Absorbance at a wavelength of 450 nm was measured to assay for protein expression levels and the protein concentration was then adjusted. The results represent at three independent experiments.
Seno K., Munakata Y., Sano M., Kawahara-Miki R., Takahashi H., Ohkuchi A., Iwata H., Kuwayama T, & Shirasuna K. (2018). Aggregation of Human Trophoblast Cells into Three-Dimensional Culture System Enhances Anti-Inflammatory Characteristics through Cytoskeleton Regulation. International Journal of Molecular Sciences, 19(8), 2322.
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