Based on our homogeneous and robust HTRF assays, the phospho-IKKβ kit is designed for detecting and studying 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.
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.
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.
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.
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.
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.
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.
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
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Phospho-IKKB Assay kit - 500 tests | 64KKBPEG | ||
| Phospho-IKKB Assay kit - 10,000 tests | 64KKBPEH | ||
| Phospho-IKKB Assay kit - 50,000 tests | 64KKBPEI |
| Description | Cat. no | Product insert | MSDS |
|---|---|---|---|
| Cellular kinase blocking reagent - 2 ml | 64KB1AAC | - | |
| Cellular kinase blocking reagent - 6 ml | 64KB1AAD | - | |
| Phospho IKKB control lysate | 64KKBTDA | - | - |
| Cellular kinase lysis buffer - 130 mL | 64KL1FDF | - |
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Investigating kinase activity in a cellular context
Translating academic research into early stage drug discovery projects
