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FACE™ NFκB p65 Profiler

in-cell Western analysis for phospho NFκB p65 (S468 and S536)

Fast Activated Cell-based ELISA (FACE™) Kits provide a simple, sensitive method for detecting protein phosphorylation directly in the cell, without making extracts or performing electrophoresis and membrane blotting. These 96-well, high-throughput assays are available in both colorimetric and chemiluminescent formats for over 20 different targets (see list at right). For complete details, click the FACE™ Method tab below.

FACE NFκB p65 Profiler Kits provide 96 rxns each of 3 antibodies that enable you to monitor and compare the levels of NFκB p65 phosphorylated at two different sites, as well as total NFκB p65. The phospho-NFκB p65 (S468) antibody specifically recognizes NFκB p65 when phosphorylated at Ser468, while phospho-NFκB p65 (S536) antibody specifically recognizes NFκB p65 that is phosphorylated at Ser536. The total-NFκB p65 antibody recognizes NFκB p65 regardless of its phosphorylation state. Click the NFκB p65 Info tab below for data and more information.

 
Name Format Cat No. Price  
FACE™ NFκB p65 Profiler 3 x 96 rxns 48300 $645 Buy Now
FACE™ NFκB p65 Profiler Chemi 3 x 96 rxns 48400 $695 Buy Now
Measurement of phosphorylated NFkB p65 (S468 and S536) and total NFkB p65 using the FACE NFkB p65 Profiler Cell Based ELISA (In Cell Western) method.
 
Figure 1: Measurement of phosphorylated and total NFκB p65.

HeLa cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 20 ng/ml TNF-α and 50 nM Calyculin A for 15 minutes and fixed. Total and phospho NFκB p65 were each assayed in triplicate using the phospho and total NFκB p65 antibodies included in the FACE NFκB p65 Profiler Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-NFκB p65 (S468) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 468 of human NFκB p65 and does not cross-react with other sites. The phospho-NFκB p65 (S536) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 536 of human NFκB p65 and does not cross-react with other sites. The total-NFκB p65 antibody recognizes NFκB p65 proteins regardless of the phosphorylation state.

NFκB p65 Overview

The transcription factor NFκB (NF-kappa-B p65, RelA, nuclear factor κB) is a key component for the inducible expression of a wide variety of cellular and viral genes. NFκB is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitory proteins, the IκBs. During the phosphorylation and degradation of IκBs, NFκB p65 is phosphorylated on multiple resides, each triggered by different stimuli but essential for maintaining NFκB transcriptional activation. Because of its role in regulating inflammatory and immune responses, high-throughput study methods for monitoring NFκB phosphorylation are in high demand.

The FACE™ Method

In FACE, cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are fixed rapidly, which preserves activation-specific protein modifications. Each well is then incubated with a primary antibody specific for the activated protein of interest. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides a colorimetric or chemiluminescent readout that is quantitative and reproducible (Figure 1). The number of cells in each well can be normalized easily with the provided Crystal Violet solution. FACE Kits also contain primary antibody specific for the native inactive protein, so you can monitor both native and activated protein levels in the same experiment. FACE eliminates cellular extractions, radioactive kinase assays, time-consuming Westerns and inefficient epitope interactions that occur on membranes. FACE is a highly sensitive high-throughput assay designed for detecting activated proteins within mammalian cells.

 
Flow chart of the FACE in cell Western method that uses a cell based ELISA to measure the levels of the native and phospho forms of signaling proteins and kinases that are activated by phosphorylation.
 
Figure 1: Flow chart of the FACE process.

Flow chart of the FACE in cell Western method that uses a cell based ELISA to measure the levels of the native and phospho forms of signaling proteins and kinases that are activated by phosphorylation.

Contents & Storage

Three 96-well plates for culturing cells, 96 rxns each of three primary antibodies (2 phospho-specific, 1 specific for native protein), HRP-conjugated secondary antibody, Quenching Solution, 1X Antibody Blocking Buffer, 1X Antibody Dilution Buffer, 10X PBS, 10% Triton X-100, 1% SDS Solution, Developing and Stop Solutions, and Crystal Violet Cell Quantification Solution. Storage conditions vary from room temperature to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.