Phospho-IKKβ (Ser177/181) & Total IKKβ Cellular Assay Kit

HTRF® cellular assay kit for measuring total and phosphorylated IKKβ directly in cells

Based on our homogeneous and robust HTRF assays, the total IKKβ and phospho-IKKβ kits are designed for detecting and studying total IKKβ or activated IKKβ when phosphorylated at Ser177 and 181 directly in whole cells. Using a streamlined protocol, amenable to low-volume format, this kit can be used from basic research to High Throughput drug screening.

   Check our lysis buffer compatibility      Check our species compatibility

Assay Principle

The assay can be run with cell lysates or using whole cells.

Upon activation, IKKβ is phosphorylated on Ser177 and 181 and after the lysis of the cell membrane, phospho- IKKβ can be detected using the kit reagents. The assays are based on sandwich immunoassays, each involving two monoclonal antibodies: the anti-phospho IKKβ antibody labeled with Eu3+-cryptate and the anti-IKKβ antibody labeled with d2. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.

The assays can be run under a two-plate protocol. They can also be further streamlined to a one-step assay.

Two-plate assay protocol

Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated IKKβ by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored.

One-plate assay protocol

Detection of phosphorylated IKKβ with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, HTS designed, enables miniaturization while maintaining HTRF quality.

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

Activation of the NF-κB is initiated by the signal-induced degradation of IκB proteins. This occurs primarily via activation of a kinase called the IκB kinase (IKK). IKK is composed of a heterotrimer of 3 subunits, IKKα and IKKβ (the two catalytic subunits) and IKKγ/NEMO (a regulatory component). Activated IKK-β phosphorylates a protein called the inhibitor of NF-κB, IκB (IκBα), which binds NF-κB to inhibit its function. Phosphorylated IκB is degraded via the ubiquitination pathway, freeing NF-κB and allowing its entry into the nucleus of the cell, where it activates various genes involved in inflammation and other immune responses. IKK-β plays a significant role in brain cells following a stroke.

Product Performance

1. Western Blot versus HTRF assay

HeLa cells were grown in a T175 flask 37°C, 5% Co2, 2 days. Stimulation was done with IL1b 0.5nM for 15min. After elimination of cell culture medium, 3ml of supplemented lysis buffer was added and incubated for 45min. Soluble supernatants were collected after 10min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF. HTRF assay shows better sensitivity than Western Blot: 12,000 cells for HTRF compared to 46,000 cells for WB.

2. TNFα dose-response on HeLa cells

Two concentrations of HeLa cells (100 and 200K cells/well) were incubated for 15 minutes at 37°C with various concentrations of TNFα. After a 30 minute lysis incubation time, phosphorylated IKKβ was measured using the two-plate assay protocol.

3. IL1β dose-response on HeLa cells

Two concentrations of HeLa cells (100 and 200K cells/well) were incubated for 15 minutes at 37°C with various concentrations of IL1β. After a 30 minute lysis incubation time, phosphorylated IKKβ was measured using the two-plate assay protocol.

4. Detection phospho and total IKKbeta on HeLa cells

Different cell densities (200K and 100K) of HeLa cells were plated under 100µL in 96-well plate and incubated overnight. Media was aspirated and 50µL of different concentrations of IL-1beta was added during 15 minutes. After incubation, media was aspirated and cells were lysed with 50µL of lysis buffer 1X for 30 min at RT under gentle shaking. 16µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho IKKbeta (Ser177/181) or total IKKbeta detection reagents were added. The HTRF signal was recorded after a 2 hour incubation at room temperature.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
IKKB total kit - 500 tests63ADK097PEG
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IKKB total kit - 10,000 tests63ADK097PEH
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IKKB total kit - 50,000 tests63ADK097PEY
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IKKB phospho-S177/181 kit - 500 tests64KKBPEG
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IKKB phospho-S177/181 kit - 10,000 tests64KKBPEH
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IKKB phospho-S177/181 kit - 50,000 tests64KKBPEY
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Companion products

DescriptionCat. noProduct insertMSDS
IKKB total kit control lysate63ADK097TDA
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Phospho-total protein blocking reagent - 2 ml64KB1AAC
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Phospho-total protein blocking reagent - 6 ml64KB1AAD
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IKKB phospho-S177/181 kit control lysate64KKBTDA
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Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
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Documents