Phospho-IKKα (Ser176/180) assay principle

The Phospho-IKKα (Ser176/180) assay measures IKKα when phosphorylated at Ser176 and Ser180. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-IKKα (Ser176/180) assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Phospho-IKKα (Ser176/180) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before adding Phospho-IKKα (Ser176/180) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-IKKα (Ser176/180) one-plate assay protocol

Detection of Phosphorylated IKKα (Ser176/180) 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.

Activation of Phospho-IKKα (Ser176/180) measured on HeLa cells

HeLa cells (Immortalized Human cervical cancer cell line) were seeded in a 96-well culture-treated plate at 100,000 cells/well for 24 hours in complete culture medium for adhesion. The cell culture medium was then removed, and the cells were treated with increasing concentrations of IL1β co-incubated with a fixed concentration of Calyculin A at 100 nM for 15 minutes at 37° C, 5% CO2, in cell culture medium containing 10% FCS. After this treatment, the cell culture medium was removed and 50 µl of supplemented Lysis Buffer#1 (1X) were dispensed into each well, followed by 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total IKKα or Phospho-IKKα (Ser176/180) detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

Cytokines including IL-1β have been described for their activating effect on IKK complex and phosphorylation of Phospho-IKKα (Ser176/180). As expected, the results obtained showed a clear dose-dependent activation of IKKα phosphorylation at Ser176 and Ser180 upon treatment with IL-1β, while the total IKKα protein expression level remained constant.

Inhibitor characterization with Phospho-IKKα (Ser176/180) and total IKKα on HeLa cells

HeLa cells were seeded in a 96-well culture-treated plate at 100,000 cells/well in complete culture medium and incubated for 24 hours at 37°C,5%CO2. Cells were treated with a dose-response of 5Z-7-Oxozeaenol (a TAK1 inhibitor) for 1 hour at 37 °C, 5% CO2. They were then co-incubated with 2nM IL1β and 100 nM Calyculin A in cell culture medium for 15 minutes at 37° C, 5% CO2. Cells were next lysed with 50 µl of supplemented Lysis Buffer#1 (1X) for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total IKKα or Phospho-IKKα (Ser176/180) detection antibodies were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, the inhibitor induced a dose-dependent decrease in IKKα (Ser176/180) phosphorylation, without any effect on the expression level of the total IKKα protein.

Phospho-IKKα assay specificity validation

Phospho-IKKα (Ser176/180) levels were assessed with the HTRF Human Phospho-IKKα (Ser176/180) kit in HAP1 cells (WT) and different HAP1 cell lines Knocked-Out for IKKα or IKKβ. Cell density was optimized beforehand to ensure HTRF detection within the dynamic range of the kit (data not shown).

The different cell lines were cultured in a 96-well plate (200,000 cells/well) for 24 hours at 37°C, 5% CO2, followed by stimulation with 5nM TNFα and 100 nM Calyculin A for 15 minutes at 37°C, 5% CO2. The cells were then lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking, and 16 µL of cell lysate were transferred into a low volume white microplate followed by 4 µL of premixed detection reagents. The HTRF signal was recorded after an overnight incubation at RT.

In HAP1 KO IKKα cells, the HTRF signal was equivalent to the non-specific signal (dotted line), indicating a complete IKKα gene silencing, whereas the IKKα level was well detected in the other cell line, as expected.

Note that similar expression levels for IKKα in WT and KO IKKβ cell lines demonstrate the selectivity of the HTRF IKKα kit over IKKβ, despite these two proteins sharing more than 50% sequence homology.

Catalog cell line references (Horizon Discovery): HAP1 Wt #C631; HAP1 KO IKKα # HZGHC000003c011; HAP1 KO IKKβ # HZGHC023808c011

HTRF Phospho-IKKA p-S176/180 assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 24h incubation, the cells were first stimulated with 2nM IL1β and 100 nM Calyculin A for 15 minutes at 37°C, 5% CO2, then lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of Phospho-IKKα (Ser176/180) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

The side-by-side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 4-fold more sensitive than the Western Blot, at least under these experimental conditions.

IKKA signaling pathway

The NF-κB pathways are involved in the regulation of the immune response, cellular growth and apoptosis. These pathways can be activated through either the classical pathway or the alternative pathway. Activation of the classical pathway leads to the stimulation of different kinases including TAK1, which activates the IκB kinase complex (IKK) via phosphorylation of IKKβ (Ser177/Ser181) or IKKα (Ser176/Ser180) subunits. Upon stimulation, the IKK complex phosphorylates IκBα, enabling p50-p65 to translocate to the nucleus. The activation of the alternative pathway leads to NIK activation and subsequent phosphorylation of the IKKα subunit.