Recombinant p65

Manufactured by Active Motif

Recombinant p65 is a purified, recombinant protein that corresponds to the p65 subunit of the NF-κB transcription factor complex. It can be used as a standard or control in various experimental applications.

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Lab products found in correlation

Transam nf κb p65 transcription factor assay kit, by Active Motif (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Recombinant mouse tnf α, by R&D Systems (1 mentions)

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Recombinant NFκB p65 protein (3 citations) Recombinant p65 protein (2 citations)

2 protocols using recombinant p65

NF-kB Activation Measurement in BV2 Cells

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BV2, BV2-Hsp72 and BV2-LacZ cells were seeded at 4×10⁵ cells per well in six well plates 36 hrs prior to treatment with 10 ng/ml recombinant mouse TNFα (R&D Systems, Minneapolis, MN). Cells were then harvested for protein at the indicated times with Phosphosafe Extraction buffer (Novagen, Darmstadt, Germany) supplemented with 0.01 volume Protease Inhibitor cocktail (Sigma, St. Louis, MO) and 5 mM DTT before use. Protein concentration was measured using the Coomassie Plus assay (Pierce, Rockford, IL). 25 µg total protein/sample was transferred to a pre-chilled Eppendorf tube and brought to 25 µl with complete lysis buffer. Aliquots were stored at −80°C until use for activated NF-κB p65 measurement. Active NF-κB was measured using the Trans AM NFκB p65 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA, cat#40096) according to the manufacturer's instructions on 20 ug total protein/sample. Three cultures were assayed for each group. Standards were prepared from recombinant p65 (Active Motif).

Sheppard P.W., Sun X., Khammash M, & Giffard R.G. (2014). Overexpression of Heat Shock Protein 72 Attenuates NF-κB Activation Using a Combination of Regulatory Mechanisms in Microglia. PLoS Computational Biology, 10(2), e1003471.

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Quantifying NF-κB Activation in PBMCs

Cited 1 time

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Inflammatory signaling via activated NF-κBin peripheral blood mononuclear cells was quantified after venipuncture between 8:30 and 11:30 AM.^{34 (link)} The mean NF-κB concentrations from triplicate determinations were expressed as nanograms of p65 per microgram of total protein using recombinant p65 (ActiveMotif) as the reference standard. The detectable range of the assay was 0.08 to 5.00 ng of NF-κB p65 per microgram of total protein. The NF-κB activation in peripheral blood mononuclear cells is a precursor to increased concentrations of inflammatory markers in plasma, and sleep disruption increases NF-κB activation.^{49 (link),50 (link)}

Black D.S., O’Reilly G.A., Olmstead R., Breen E.C, & Irwin M.R. (2015). Mindfulness Meditation and Improvement in Sleep Quality and Daytime Impairment Among Older Adults With Sleep Disturbances: A Randomized Clinical Trial. JAMA internal medicine, 175(4), 494-501.

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