Histone H1 is a linker histone that binds to nucleosomes and linker DNA to stabilize higher-order chromatin structure and regulate DNA accessibility. H1 binds to the exterior of the nucleosome, compacting chromatin into 30-nm fibers and higher-order structures. Mammals express multiple H1 variants with distinct expression patterns and regulatory functions. H1 binding is dynamically regulated through post-translational modifications that modulate chromatin compaction and gene expression. Altered H1 expression and mutations are found in various cancers and developmental disorders, affecting genome stability and transcriptional regulation.
The AlphaLISA SureFire Ultra Human Phospho-Histone H1 (Thr17) is a sandwich immunoassay for the quantitative detection of phospho-Histone H1 (Thr17) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
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 enables cell 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 robust 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 Hydroxyurea
HeLa and 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 hydroxyurea 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 hydroxyurea resulted in a dose-dependent decrease 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.
