AlphaLISA SureFire Ultra Human Total IκKβ Detection Kit, 500 Assay Points

Total-AlphaLISA SureFire Ultra two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.

Induction of IKKb phosphorylation in TNFα treated cells

A549 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of TNFα for 10 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IKKβ Total and Phospho (Ser177/181) levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, TNFα triggered a dose-dependent increase in the levels of Phospho (Ser177/181) IKKβ while Total levels remained unchanged.

Specificity of the Phospho IKKβ assay

Specificity of the Total IKKβ assay was assessed by evaluating recombinant IKKα (Abcam, ab102103) and IKKβ (Millipore, 14-485) active recombinant proteins.

Both proteins were prepared at 15 ng/ml in Lysis Buffer, and IKKβ Total signal was evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of protein was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

IKKβ Total signal was only detected with IKKβ recombinant protein demonstrating assay specificity.

Expression of IKKβ in various cell lines

Adherent cells were grown to confluency in a T175 flask complete medium and were lysed with Lysis Buffer at a density of 1 x 10⁶ cells/mL. Suspension cells were harvested then washed in HBSS and lysed with Lysis Buffer at a density of 3.2 x 10⁶ cells/mL.

IKKβ Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (5,000 adherent and 15,000 suspension) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, IKKβ is expressed in a wide range of cell types.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from Karpas 299 cells resuspended in HBSS and treated with 2 ng/mL IL-1β for 5 minutes and lysed with 5X Lysis Buffer at a final cell density of 8 x 10⁶ cells/mL for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and IKKβ Total and Phospho (S177/181) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect IKKβ expression in less than 2,500 cells/datapoint.