Alpha-Tubulin Housekeeping Cellular Kit
Total NFkB cellular kit HTRF®
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.
See more
-
Ready-to-use
-
High sensitivity
-
Faster and more convenient than ELISA
-
No-wash
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 proteins expression under a no-wash assay format.
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 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.
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.
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.
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.
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.
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.
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
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF and Alpha solutions for NASH - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
HTRF assays for Oncology and Inflammation
Signaling in the Immune System - Brochures
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
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
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
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
A single technology for 2D cells, 3D cells, and xenograft models - 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
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
STING HTRF offer to bridge innate and adaptive immunity
cGAS-STING signaling pathway from A to Z - Brochures
Manual NFkB total Kit / 64NFTPEG-64NFTPEH
64NFTPEG-64NFTPEH - Product Insert
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
HTRF HMGB1, IL-1β and IL-18 assays discriminate between inflammasome triggered pyroptosis, and necroptosis - Application Notes
Neuroinflammation study combining hiPSC-derived astrocytes and HTRF
See published experiments and data demonstrating how immunoassays rise to the challenge of astrocyte studies in neuroinflammation research - Application Notes
On-demand webinar: Linking Neuroinflammation and Neurodegeneration
New insight into neuroinflammation research - Videos
Assays for autoimmunity research
Advance your research on autoimmune diseases - Flyers
Assays for neurosciences research
Advance your research on neurodegenerative diseases - Flyers
Advance your research on Fibrosis
Kits and reagents for Fibrosis research - Flyers
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Assessment of drug efficacy and toxicity by combining innovative technologies
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Plate Reader Requirement
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
Let's find your reader
You might be interested in