AlphaLISA SureFire Ultra Human Phospho-Histone H1 (Thr17) Detection Kit, 10,000 Assay Points

Phospho-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 Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Phospho-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Phosphorylated 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 AlphaLISA SureFire Ultra quality.

Nocodazole induces Histone H1 phosphorylation in a dose-dependent manner

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 Nocodazole for 20 hours.

After treatment, cells were lysed with 200 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). Histone H1 Phospho (Thr17) and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, Nocodazole triggered a dose-dependent increase in the levels of Phospho Histone H1 (Thr17) with no changes observed in Total ERK levels.

Induction of Histone H1 phosphorylation by colcemid

HeLa 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 colcemid for 20 hours.

After treatment, the cells were lysed with 200 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). Histone H1 Phospho (Thr17) and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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, Colcemid triggered a dose-dependent increase in the levels of Phospho Histone H1 (Thr17) with no changes observed in Total ERK levels.

Inhibition of Histone H1 phosphorylation with palbociclib

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 palbociclib at increasing concentrations for 20 hours.

After treatment, the cells were lysed with 200 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). Histone H1 Phospho (Thr17) and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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.

Treatment with palbociclib resulted in a dose-dependent decrease in the levels of Phospho Histone H1 (Thr17) with no changes observed in Total ERK levels.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HEK293 cells cultured to confluence in a T175 flask and lysed in 40 mL of Lysis Buffer B for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and Histone H1 Phospho (Thr17) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect Histone H1 (Thr17) in HEK293 cells in less than 200 cells/datapoint.