The total IKK-beta kit monitors the cellular IKK-beta expression level and can be used as a normalization assay for the phospho-IKK-beta kit.
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  • Ready-to-use Ready-to-use
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The total IKK-beta kit monitors the cellular IKK-beta expression level and can be used as a normalization assay for the phospho-IKK-beta kit.
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Overview

The total-IKK-beta assay enables the detection of total IKK-beta directly in whole cells. Using a streamlined protocol, amenable to low-volume formats, this kit can be used from basic research to High Throughput drug screening.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-IKKß assay principle

The Total IKK-beta assay quantifies the expression level of IKK-beta in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total IKK-beta assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of IKKß in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total IKKß cellular assay principle

Total-IKKß 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 Total IKK-beta HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total IKKß cellular assay 2-plate assay protocol

Total-IKKß cellular 1-plate assay protocol

Detection of total IKK-beta cellular 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.
Total IKKß cellular 1-plate assay protocol

Detection of phospho and total IKK-beta on HeLa cells

Different cell densities (200K and 100K) of HeLa cells were plated under 100 µL in 96-well plate and incubated overnight. Media was aspirated and 50 µL of different concentrations of IL-1 beta was added during 15 minutes. After incubation, media was aspirated and cells were lysed with 50 µL of lysis buffer 1X 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 HTRF phospho IKK-beta (Ser177/181) or total IKK-beta detection reagents were added. The HTRF signal was recorded after a 2 hour incubation at room temperature.
Detection phospho and total IKK-beta on HeLa cells

IKKß Simplified Pathway

Activation of the NF-?B is initiated by the signal-induced degradation of I?B proteins. This occurs primarily via activation of a kinase called the I?B kinase (IKK). IKK is composed of a heterotrimer of 3 subunits, IKK-alpha and IKK-beta (the two catalytic subunits) and IKK-gamma/NEMO (a regulatory component). Activated IKK-beta phosphorylates a protein called the inhibitor of NF-?B, I?B (I?Ba), which binds NF-?B to inhibit its function. Phosphorylated I?B is degraded via the ubiquitination pathway, freeing NF-?B and allowing its entry into the nucleus of the cell, where it activates various genes involved in inflammation and other immune responses. IKK-beta plays a significant role in brain cells following a stroke.
IKK-beta signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Translating academic research into early stage drug discovery projects

In collaboration with European ScreeningPort - Scientific Presentations

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

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 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

Product Insert IKK b total Kit / 63ADK097PEG-63ADK097PEH

63ADK097PEG-63ADK097PEH - 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

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

Characterize STING agonists with HTRF human IFNb

Data about HTRF IFNb correlation with gene reporter assay and ELISA - Application Notes

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

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