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HTRF Human and Mouse Phospho-NF-κB (Ser536) Detection Kit, 50,000 Assay Points

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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Feature	Specification
Application	細胞シグナル伝達
Sample Volume	16 µL

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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Product variants

Unit Size: 96 assay points

Part #:

64NFBPET

Unit Size: 500 assay points

Part #:

64NFBPEG

Unit Size: 10,000 assay points

Part #:

64NFBPEH

Unit Size: 50,000 assay points

Part #:

64NFBPEY

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).

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Product information

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.

How it works

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 proteins phosphorylation state under a no-wash assay format.

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) 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.

Assay validation

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.

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.

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

HeLa cells were plated and cultured for 24h before being exposed to increasing 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.

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.

Simplified pathway

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.

Specifications

Other specifications

Application	Cell Signaling
Brand	HTRF
Detection Modality	HTRF
Molecular Modification	Phosphorylation
Product Group	Kit
Sample Volume	16 µL
Shipping Conditions	Shipped in Dry Ice
Target Class	Phosphoproteins
Target Species	Human Mouse
Technology	TR-FRET
Therapeutic Area	NASH/Fibrosis Neuroscience Oncology & Inflammation
Unit Size	50,000 assay points