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AlphaLISA SureFire Biotin-Free Human and Mouse Total Histone H3 Detection Kit, 100 Assay Points
AlphaLISA SureFire Biotin-Free Human and Mouse Total Histone H3 Detection Kit, 100 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total Histone H3 assay is a sandwich immunoassay for quantitative detection of total Histone H3 in cellular lysates using Alpha Technology.
| Feature | Specification |
|---|---|
| Application | 細胞シグナル伝達 |
| Protocol Time | 2h at RT |
| Sample Volume | 30 µL |
The AlphaLISA™ SureFire® Biotin-Free Human and Mouse Total Histone H3 assay is a sandwich immunoassay for quantitative detection of total Histone H3 in cellular lysates using Alpha Technology.
Product variants
Unit Size: 100 Assay Points
Part #:
ASBF-THISH3-A-HV
Unit Size: 500 Assay Points
Part #:
ASBF-THISH3-A500
Unit Size: 10,000 Assay Points
Part #:
ASBF-THISH3-A10K
Unit Size: 50,000 Assay Points
Part #:
ASBF-THISH3-A50K
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).
AlphaLISA SureFire Biotin-Free Human and Mouse Total Histone H3 Detection Kit, 100 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
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Product information
Overview
Histone H3 is a core component of the nucleosome, the fundamental repeating unit of chromatin, in which two copies of H3 pair with H4, H2A, and H2B to form the histone octamer around which approximately 147 base pairs of DNA are wrapped. Histone H3 serves as a major platform for post-translational modifications (PTMs) including methylation, acetylation, phosphorylation, and ubiquitination at its N-terminal tail, which collectively constitute a histone code that regulates chromatin accessibility and gene expression. Key H3 modifications include H3K4me3 (active promoters), H3K27me3 (Polycomb-mediated repression), H3K9me3 (constitutive heterochromatin), H3K27ac (active enhancers), and H3K36me3 (transcribed gene bodies). Histone H3 variants including H3.1, H3.2, H3.3, and the centromeric variant CENP-A have distinct deposition mechanisms and genomic distributions that contribute to specialized chromatin states. Mutations in histone H3 — particularly H3K27M and H3G34R/V — are oncogenic drivers in pediatric brain tumors, acting as dominant inhibitors of PRC2 and other chromatin-modifying enzymes. Total histone H3 is widely used as a chromatin loading control in histone modification studies and as a reference for normalizing PTM-specific signals.
The AlphaLISA SureFire Biotin-Free Human and Mouse Total Histone H3 Detection Kit is a sandwich immunoassay for the quantitative detection of total histone H3 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 Biotin Free kits are compatible with:
- Cell and tissue lysates
- Antibody modulators
- Biotherapeutic antibodies
- Biotin rich samples
AlphaLISA SureFire Biotin Free kits can be used for:
- Cellular kinase assays
- Receptor activation studies
- High-throughput screening for preclinical studies
How it works
Total-AlphaLISA SureFire Biotin Free assay principle
The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Biotin Free assay uses two antibodies which recognize two different distal epitopes 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 a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with the CaptSure3 tag. In the presence of target 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 protein present in the sample.
Total-AlphaLISA SureFire Biotin Free 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 Biotin Free 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 Biotin Free one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Biotin Free 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 quality.
Assay validation
Validation of Histone H3 Total in cells treated with Trichostatin A
HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of Trichostatin A (TSA) for 4 hours.
After treatment, the cells were lysed with 200 µL of Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). Lysate was further diluted using Lysis Buffer B and evaluated for Acetyl Histone H3 (K27) and Histone H3 Total levels using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 100 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, TSA treatment resulted in an increase of Acetyl Histone H3 (K27) levels, while Total levels remained unchanged.
Validation of Histone H3 Total in cells treated with A485
HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of A485 for 3 hours.
After treatment, the cells were lysed with 200 µL of Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). Acetyl Histone H3 (K27) and Histone H3 Total levels were evaluated using AlphaLISA SureFire Biotin Free 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, A485 (p300/CREBBP inhibitor) treatment resulted in a decrease of Acetyl Histone H3 (K27) levels, while Total levels remained unchanged.
Karpas 299 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and treated with increasing concentrations of A485 for 3 hours.
After treatment, the cells were lysed with the addition of 50 µL 5X Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). Acetyl Histone H3 (K27) and Histone H3 Total levels were evaluated using AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 16,000 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, A485 (p300/CREBBP inhibitor) treatment resulted in a decrease in levels of Acetyl Histone H3 (K27), while Total levels remained unchanged.
Validation of Histone H3 Total in cells treated with dCBP-1
HeLa cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of dCBP-1 for 2 hours.
After treatment, the cells were lysed with 200 µL of Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). Acetyl Histone H3 (K27) and Histone H3 Total levels were evaluated using AlphaLISA SureFire Biotin Free 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, dCBP-1 (p300/CREBBP PROTAC) treatment resulted in a decrease in levels of Acetyl Histone H3 (K27), while Total levels remained unchanged.
Assay versatility
Histone H3 expression in various cell lines
Adherent cells were seeded in a 6-well plate (500,000 cells/well) in complete medium incubated overnight at 37°C, 5% CO2. Suspension cells were harvested and seeded in a 96-well plate (400,000 cells/well) in complete medium.
Cells were lysed with Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). Lysate was further diluted and Histone H3 Total levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 500 adherent cells and 1,000 suspension cells) 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.
Histone H3 Total shows very high basal expression in a wide variety of cell lines.
Histone H3 Total expression in human PBMCs
Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors using Ficoll Plaque Plus (Merck GE17-1440-02). PBMCs were lysed at 4 x 106 cells/mL with Lysis Buffer B for 30 minutes at RT with shaking. Lysate was serially dilute and Histone H3 Total levels were evaluated using the AlphaLISA SureFire Biotin Free assay.
For the detection step, 10 µL of cell lysate 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.
Histone H3 Total shows basal expression in PBMCs. Dotted line represents assay background.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Biotin-Free
|
| Detection Modality |
Alpha
|
| Product Group |
Kit
|
| Protocol Time |
2h at RT
|
| Sample Volume |
30 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
Histone H3
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
Mouse
|
| Technology |
Alpha
|
| Therapeutic Area |
Oncology
|
| Unit Size |
100 Assay Points
|
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