Phospho-NFkB (Ser536) & Total NFkB Cellular Assay Kits

Phospho- and total NFkB detection as a readout of the NFkB pathway

Based on our homogeneous and robust HTRF® technology, the phospho-NFkB (Ser536) kit enables the detection of NKkB p65 (Nuclear factor kappa B) phosphorylated at Serine 536 while the total NFkB kit is designed for the detection of total NFkB p65, phosphorylated and unphosphorylated. 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. NF-κB is frequently observed in many cancers and is a key player in the inflammatory response.

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Assay Principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total NFkB assays are based on a TR-FRET sandwich immunoassay format comprising two specific monoclonal anti-YAP antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with NFkB, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-NFkB can be quantitatively detected using the HTRF phospho-NFkB(Ser536) and total NFkB cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

This protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol provides enhanced speed and simplicity, enabling all throughputs and fast results while maintaining a high-quality sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified Pathway

Regulation of the NFkB pathway

NFkB (Nuclear Factor kappa B) is a super-family with 5 members: p65 (RelA), RelB, c-Rel, p50/p105 (NF-B1), p52/p100 (NF-B2). The NF-B transcription factor consists of two subunits of either homo- or heterodimers of RelA/p65, c-Rel, and p50, and are involved in the regulation of the immune system in response to infection and cellular stress.

Two main NFkB pathways exist. The classical pathway involves p65 & p50 and is stimulated by cytokines (such as TNFa or IL1) or TLR activation (through LPS). The alternative pathway involving RelB, p52/p100 is mainly activated in lymphocyte generation.

Inactive NFkB dimers are sequestered in the cytoplasm by interaction with the kB inhibitor (IkB). Upon stimulation, the IB proteins are phosphorylated by one of a number of IB kinases (IKK-a, -b, and -g), ubiquitinylated and degraded, thereby allowing the NF-B complex to translocate into the nucleus. The active NFkB translocates into the nucleus where it mediates gene transcriptions, such as IFNγ, IL6 or TNFα and leads to biological responses such as inflammatory response, cell survival, and cellular proliferation. The phosphorylation of NFkB on residue Ser 536 has been described as enhancing its transcriptional activity (Sazaki et al, JBC 2005).

Deregulations of NFkB pathways have been found in several auto-immune disorders such as arthritis, asthmas, and multiple sclerosis, but also in some types of cancer, like leukemia, where NFkB deregulation induces anarchical cell proliferation.

Product Performance

1. Detection of phospho-NFkB (Ser536) and total NFkB in various human and mouse cell lines

Human (HeLA, Jurkat, U 937) and murine (NIH 3T3) 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 phospho-NFkB (Ser536) or total NFkB detection reagents were added. The HTRF signal was recorded after an overnight incubation.

2. HTRF assay compared to Western Blot using phospho and total NFkB assays

Human HeLa cells were grown in a T175 flask at 37 °C, 5% CO2 for 48 h, then stimulated for 10 min with 10 nM TNFalpha. After medium removal, the cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed, and analyzed side-by-side by Western Blot and by HTRF. Results show that HTRF Phospho and total NFkB cellular assays are more sensitive than the Western Blot, as 1 000 cells are sufficient for minimal signal detection when using the HTRF phospho- or total NFkB assays while 2 000 cells and 4 000 are needed for a Western Blot signal.

3. TNFalpha dose-response on HeLa cells using phospho and total NFkB cellular assays

100,000 HeLa cells were plated in 96-well plate in cell culture medium and incubated for 24h, at 37°C, 5% CO2. Increasing concentrations of TNFalpha were added for 10 min. After cell culture medium removal, cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 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.

4. Pharmacological response on Phospho-NFkB and total NFkB of BAY 11-7085, an inhibitor of IKB

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. In addition, the phosphorylation of NFKB declines with increasing BAY concentrations. Consequently the normalization signal calculated from phospho- & total NFkB level remains stable. This set of data is a good illustration of the need to analyze phospho, total and normalized values separately, in order to correctly assess the Mechanism of Action of a drug. For more information, please refer to the application note:"A guideline for HTRF® cell-based phospho-protein data normalization".

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 with 50µl of lysis buffer and lysates were transferred into a 384-well sv white microplate for detection of both HTRF phospho and total NFkB.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
NFkB phospho-S536 kit - 500 tests64NFBPEG
NFkB phospho-S536 kit - 10,000 tests64NFBPEH
NFkB phospho-S536 kit - 1 x 96 tests64NFBPET
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NFkB phospho-S536 kit - 50,000 tests64NFBPEY
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NFkB total kit - 500 tests64NFTPEG
NFkB total kit - 10,000 tests64NFTPEH
NFkB total kit - 50,000 tests64NFTPEY
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Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein lysis buffer #4 - 130 mL64KL4FDF
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NFkB phospho-S536 kit control lysate64NFBTDA
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NFkB total kit control lysate64NFTTDA
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