Total NFkB cellular kit

The total-NFkB kit detects cellular NFkB, and can be used as a normalization assay for the p-NFkB kit. It is a leading product in cancer and inflammatory research.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The total-NFkB kit detects cellular NFkB, and can be used as a normalization assay for the p-NFkB kit. It is a leading product in cancer and inflammatory research.
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Overview

Total NFkB kit serves as a normalization assay with our Phospho-NFkB kits. NFkB (Nuclear Factor Kappa B) is of great interest in cancer research and a key player in the inflammatory response. The buffers of both HTRF phospho- and total NFkB assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-NFkB assay principle

The Total-NFkB assay quantifies the expression level of NFkB in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-NFkB assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of NFkB in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total NFkB assay principle

Total-NFkB 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total NFkB HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total NFkB 2-plate assay protocol

Total-NFkB 1-plate assay protocol

Detection of total NFkB with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Total NFkB 1-plate assay protocol

Detection of total NFkB in various human/mouse cells

Human and murine cells in serum-deprived cell culture medium were plated at 40,000 cells per well in a 96-well plate and incubated for 24h at 37°C, 5% CO2. The phosphorylation state was induced by a 10 min stimulation time with 10 nM TNFalpha or 2 nM IL1beta. After stimulation, medium was removed and cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into 384-well sv white microplate, and 4 µL of the HTRF total NFkB detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Detection of total NFkB in various mouse cells

HTRF assay vs WB using total-NFkB assay

Human HeLa cells were cultured for 48 followed by TNFalpha stimualation. Following lysis, soluble fractions were collected after centrifugation. Serial dilutions of the cell lysate were analyzed side-by-side by Western Blot and by HTRF. Results show that HTRF total-NFkB cellular assays is more sensitive than the Western Blot, as 1 000 cells are sufficient for minimal signal detection when using the HTRF total-NFkB assay while 4 000 are needed for a Western Blot signal.
HTRF assay vs WB using total NFkB assays

TNFa dose-response on HeLa cells using phospho and total NFkB kit

HeLa cells were plated and cultured for 24h before being exposed to ncreasing concentrations of TNFalpha. Following cell lysis, 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the phospho-NFkB (Ser536) or total NFkB detection reagents were added. The HTRF signal was recorded after an overnight incubation. Stimulation with increasing concentration of TNFa induces phosphorylation of NFkB, while the total amount of protein, unphosphorylated and phosphorylated, remains stable.
TNFa dose-response on HeLa cells using phospho-NFkB kit
TNFa dose-response on HeLa cells using total NFkB kit

Pharmacological response on Phospho and total NFkB of BAY 11-7085

Prolonged exposure to BAY 11-7085 induces a downregulation of the NFkB expression level, as shown by the decrease of the HTRF total NFKB signal. Phosphorylation of NFKB also declines with increasing BAY concentrations. Consequently, the normalization signal calculated from phospho- & total NFkB level remains stable. 40,000 cells of the U937 cell line were stimulated by increasing concentrations of BAY 11-7085 for a 3 hours, and co-stimulated for 10 min with 10 nM TNFalpha. Cells were lysed and transferred into a 384-well sv white microplate for detection of both HTRF phospho and total NFkB.
Pharmacological response on Phospho-NFkB p65 Ser536 of BAY 11-7085
Pharmacological response from total NFkB p65
Pharmacological response on Phospho and total NFkB of BAY 11-7085

Regulation of the NFkB pathway

NFkB is in a super-family with 5 members and consists of two subunits of either homo- or heterodimers that are involved in the regulation immune respose. Two main NFkB pathways exist. The classical pathway involves p65 & p50 and is stimulated by cytokines or TLR activation. The alternative pathway is mainly activated in lymphocyte generation. Inactive NFkB dimers are sequestered in the cytoplasm. Upon stimulation, the IB proteins are phosphorylated, ubiquitinylated and degraded, which activates the NF-B complex, causing it to translocate into the nucleus. Activated NFkB helps mediate gene expression, inflammatory response, cell survival and cellular proliferation. Deregulation of NFkB pathways have been found in several auto-immune disorders but also in some types of cancer.
NFkB signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

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Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

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Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

Product Insert NFkB total Kit / 64NFTPEG-64NFTPEH

64NFTPEG-64NFTPEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Novel HTRF platform to delineate STING pathway

Explore the whole STING pathway with a single technology - Posters

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

STING, the next candidate for cancer immunotherapy

Infographic about STING protein - Infographics

A Guide To Current Therapies in Immuno Oncology

Clear overview of past, present and future of immunotherapy - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Characterize STING agonists with HTRF human IFNb

Data about HTRF IFNb correlation with gene reporter assay and ELISA - Application Notes

Note about HMGB1 assay

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