Histone H3 Ac-K27 Biotin Free Assay Kit Human and Mouse

The AlphaLISA™ SureFire® Ultra™ assay enables the rapid and sensitive detection of total and phosphorylated cellular proteins. AlphaLISA™ assays utilize two bead types: Acceptor Beads and Donor Beads. The Acceptor Bead is coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody which is labeled with a CaptSure™ tag. The Donor Bead is coated with streptavidin to capture the biotinylated antibody.  

In the presence of a target protein, the two target-specific antibodies bring Donor and Acceptor Beads into close proximity. When the Donor Beads are activated by a laser (680 nm), singlet oxygen is transferred to the Acceptor Bead leading to the production of an Alpha signal. The amount of light emission (615 nm) from the Acceptor Bead is directly proportional to the amount of target protein present in the sample. If an Acceptor Bead is not in close proximity (i.e. 200 nm) of a Donor bead, little to no signal is produced over background. 

The assay can be executed in a 1-plate or 2-plate assay protocol (Refer to Manual for more details).  

1-plate assay protocol: culturing of cells, treatment, lysis and assay are performed in a single well, enabling miniaturization in high throughput screening programs. 

2-plate assay protocol: cells are cultured and treated in a 96-well culture plate and lysates are then transferred into a separate plate for assay. This format allows the evaluation of multiple targets from a single lysate. 

Specificity of the Acetyl Histone H3 (K27) assay was assessed by a peptide competition assay. Acetyl Histone H3 K4, K9, K14, K18, K23, K27 and non-acetyl peptides were serially diluted into a fixed concentration of HeLa positive control lysate. Lysates were then assayed using the Acetyl Histone H3 (K27) SureFire® Biotin Free kit.

Data obtained with a 2-plate, 2-incubation protocol. HeLa cells were seeded at 100K cells/well in a 96-well plate and treated with increasing concentrations of Trichostatin A (TSA) for 4 hours. Cells were lysed with 200 μL Lysis Buffer B for 30 minutes with shaking. Lysate was further diluted with Lysis Buffer B and assayed separately for Acetyl Histone H3 (K27) and Histone H3 Total using respective SureFire® Biotin Free kits. Equivalent to approximately 100 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. Karpas 299 cells were seeded at 400K cells/well in a 96-well plate and treated with increasing concentrations of A485 for 4 hours. Cells were lysed with the addition of 50 μL of 5X Lysis Buffer Lysis Buffer B for 30 minutes with shaking. Lysate was assayed separately for Acetyl Histone H3 (K27) and Histone H3 Total using respective SureFire® Biotin Free kits. Equivalent to approximately 16,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. HeLa cells were seeded at 40K cells/well in a 96-well plate and treated with increasing concentrations of dCBP-1 for 2 hours. Cells were lysed with 200 μL of Lysis Buffer Lysis Buffer B for 30 minutes with shaking and assayed separately for Acetyl Histone H3 (K27) and Histone H3 Total using respective SureFire® Biotin Free kits. Equivalent to approximately 2,000 cells/datapoint.

Data obtained from measurement of Acetyl Histone H3 (K27) in various cell types treated with 500 ng/mL Trichostatin A for 4 hours and lysed with Lysis Buffer B for 30 minutes with shaking. Equivalent to approximately 500 cells/datapoint for adherent cells or 1,000 cells/datapoint for suspension cells.

Data obtained from measurement of Acetyl Histone H3 (K27) in PBMCs untreated or treated with 500 ng/mL Trichostatin A for 4 hours and lysed with Lysis Buffer B for 30 minutes with shaking. Lysate was serially diluted with Lysis Buffer B and assayed using the Acetyl Histone H3 (K27) SureFire® Biotin Free kit.