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FunctionELISA™ IκBα

fast, accurate quantification of phosphorylated IκBα (S32/S36)

The FunctionELISA IκBα Kit offers a fast, simple method to monitor the phosphorylation of IκBα, an event required for NFκB translocation. The kit utilizes the Sandwich ELISA technique to capture and quantifiably measure the amount of phospho-IκBα present in a sample, making the kit useful in monitoring the activation of NFκB and the many genes it regulates. Click the IκBα Info tab below for data and more information.

FunctionELISA IκBα comes ready-to-use, with Capture Antibody specific for the phosphorylated form of IκBα already bound to the plate; this saves you the time and inconvenience of performing overnight incubations. The kit employs highly sensitive chemiluminescent detection, so requires the use of a luminometer. For more information, click the Method & Contents tabs below.

 
Name Format Cat No. Price  
FunctionELISA™ IκBα 1 x 96 rxns 48005 $565 Buy Now
5 x 96 rxns 48505 $2,275 Buy Now

FunctionELISA IκBα includes a Capture Antibody specific for IκBα that has been phosphorylated at serines 32,36. It utilizes highly sensitive chemiluminescent detection that enables accurate study of the phosphorylation state of IκBα in response to external stimuli (Figure 1). Because of the chemiluminescent detection method, the FunctionELISA IκBα Kit requires the use of a luminometer.

FunctionELISA IκBα and Western blot methods used to monitor the induction of IkBa phosphorylation
Figure 1: Induction of IκBα phosphorylation.

Jurkat cells were stimulated with 1 nM TNF-α and harvested at the indicated time points. Whole-cell lysate was assayed using Western blot analysis with Active Motif’s IκBα phospho Ser32,36 mAb (Catalog No. 40904) (top gel) and the FunctionELISA IκBα Kit (bottom graph). The FunctionELISA Kit clearly provides more quantitative results than are possible with Western.

IκBα Overview

NFκB is regulated by the IκB family of inhibitory proteins. Phosphorylation of IκBα can be triggered by a wide range of extracellular signals, including cytokines and chemokines. It leads, ultimately, to activation of NFκB. Thus, analysis of the phosphorylation state of IκBα provides insights about NFκB and the many genes it regulates. (For additional tools for the study of NFκB, see TransAM® NFκB p50, p52, p65 and Family Kits and NFκB-related antibodies).

Why use FunctionELISA?

  • Quantitative results in just hours
  • ELISA format eliminates need to run, blot and develop gels
  • Analyze multiple samples in low-volume, high-throughput experiments
  • Complete kit with all required reagents and controls
  • Ready-to-use format with capture antibody pre-coated on the plate
  • Ability to assay both cell and tissue samples

Sandwich ELISA method

FunctionELISA IκBα utilizes the Sandwich ELISA technique to capture and quantifiably measure the amount of phosphorylated IκBα present in a sample. Sandwich ELISAs use two antibodies that recognize different epitopes on the protein being studied. The first antibody is immobilized in the wells of an ELISA plate. When cell lysate is added, the specific protein of interest is bound by this Capture Antibody. A second antibody, called the Detecting Antibody, is then added. It binds to the bound protein. A horseradish peroxidase (HRP)-conjugated secondary antibody binds to the Detecting Antibody to quantify the amount of protein bound by the Capture Antibody (Figure 1).

Flow chart of the FunctionELISA method for quantifying signaling proteins.
 
Figure 1: Sandwich ELISA schematic.

The FunctionELISA IκBα Kit uses Capture and Detecting Antibodies for sensitive, accurate quantification of phosphorylated IκBα (Ag). The kit uses horseradish peroxidase (HRP)-conjugated secondary antibody that binds to the Detecting Antibody to quantify the amount of phospho-IκBα bound by the Capture Antibody.

Contents & Storage

One or five 96-well assay plates coated with Capture Antibody, Detecting Antibody, HRP-conjugated Secondary Antibody, Lysis and Wash Buffers, Protease Inhibitor, DTT, and Control protein. The FunctionELISA IκBα Kit requires the use of a luminometer.