The phospho-NFkB (Ser536) kit enables the cell-based quantitative detection of phosphorylated NFkB at Ser536 as a readout of the NFkB pathway.
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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 phospho-NFkB (Ser536) kit enables the cell-based quantitative detection of phosphorylated NFkB at Ser536 as a readout of the NFkB pathway.
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Overview

The Phospho-NFkB (Ser536) cellular assay kit is optimal for measuring phosphorylated NFkB (Nuclear Factor Kappa B) at Ser536 as a readout of the NFkB pathway. NFKB is frequently observed in many cancers, and is a key player in the inflammatory response.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-NFkB (Ser536) assay principle

The Phospho-NFkB (Ser536) assay measures NFkB when phosphorylated at Ser536. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-NFkB (Ser536) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-NFkB (Ser536) assay principle

Phospho-NFkB (Ser536) 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 Phospho-NFkB (Ser536) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-NFkB (Ser536) 2-plate assay protocol

Phospho-NFkB (Ser536) 1-plate assay protocol

Detection of Phosphorylated NFkB (Ser536) 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.
Phospho-NFkB (Ser536) 1-plate assay protocol

Detection of phospho-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 TNF alpha or 2 nM IL1 beta. 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 phospho-NFkB (Ser536) detection reagents were added. The HTRF signal was recorded after an overnight incubation.
Detection of phospho-NFkB in various human/mouse cells

HTRF assay vs WB using phospho-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 Phospho-NFkB cellular assay is more sensitive than the Western Blot, as 1 000 cells are sufficient for minimal signal detection when using the HTRF phospho-NFkB assays while 2 000 cells are needed for a Western Blot signal.
HTRF assay vs WB using phospho-NFkB assay

TNFa dose-response on HeLa cells using phospho-NFkB assay

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) detection reagents were added. The HTRF signal was recorded after an overnight incubation. Stimulation with increasing concentration of TNFa induced phosphorylation of NFkB.
TNFa dose-response on HeLa cells using phospho-NFkB assay

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

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-NFkB.
Pharmacological response on Phospho-NFkB p65 Ser536 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.
Regulation of the NFkB pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

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

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - 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

TCR signaling investigation with HTRF phospho assays

Study a pathway of interest in PBMC and T cells - 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

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® cell signaling platform combined with iCell® Hepatocytes

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

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 P-S536 Kit / 64NFBPEG-64NFBPEH

64NFBPEG-64NFBPEH - 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

A Guide To Current Therapies in Immuno Oncology

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

STING, the next candidate for cancer immunotherapy

Infographic about STING protein - Infographics

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

Note about HMGB1 assay

HTRF HMGB1, IL-1β and IL-18 assays discriminate between inflammasome triggered pyroptosis, and necroptosis - Application Notes

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