Cellomics HCS Reagent Kit for detection of phosphorylated core histone protein H2AX (Ser139). | | | | | control | treat cells | detect phospho–H2AX | analyze | conclude |
DescriptionThe Cellomics Phospho-H2AX Activation Kit contains optimized reagents for the detection and quantitation of phosphorylated H2AX (Ser139) in the nucleus. The kit contains a primary monoclonal antibody that detects only the phosphorylated form of human H2AX, a goat anti-mouse secondary antibody conjugated to DyLight 549 Fluorophore and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays. The nucleosome is made of four core histone proteins (H2A, H2B, H3 and H4). H2AX belongs to a H2A family of histones. DNA damage induction by various agents leads to rapid phosphorylation of H2AX at Ser139 (also known as Gamma H2AX) by ATM, ATR or DNA protein kinase leading to the formation of DNA foci at the site of DNA double-strand breaks (DSBs). Phosphorylated H2AX helps in recruiting the proteins responsible for double-strand break repair. Phosho-H2AX in the nucleus of A549 cells treated with etoposide was assayed using the Cellomics Phospho-H2AX Activation Kit, the Cellomics ArrayScan HCS Reader (Figure 1) and the Compartmental Analysis BioApplication Software Module. Induction of DNA damage by etoposide leads to phosphorylation of H2AX. The output parameter for this assay is the nuclear intensity of phosphor-H2AX staining after treatment. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy. | |  | Non-treated cells | Treated cells | Dose response curve | Figure 1. Staining of phospho-H2AX in A549 cells treated with vehicle (control; 0.1% DMSO in media)(left) or with 50 µM etoposide for 1 hour (center). Cells were stained according to the kit protocol and imaged using a Cellomics ArrayScan HCS Reader. Dose response curve for etoposide-treated A549 cells (right). Data represents mean +/-SD from three plates (eight wells per 96-well plate per dose of etoposide). EC50 = 68.21+/-1.25 µM. |
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Kit Contents| Component | 8402901 | 8402902 | | Phospho-H2AX Primary Antibody | 6 µl | 30 µl | DyLight 549 Conjugated
Goat Anti-Mouse IgG | 30 µl | 72 µl | | Hoechst Dye | 30 µl | 30 µl | | Wash Buffer (10X Dulbecco's PBS) | 100 ml | 100 ml | Wash Buffer II (10X Dulbecco's PBS with
1% Tween®-20) | 100 ml | 100 ml | Permeabilization Buffer (10X D-PBS with 1% Triton® X-100) | 100 ml | 100 ml | | Blocking Buffer (10X) | 85 ml | 85 ml | | Thin Plate Seal Assembly | 7/pack | 7/pack |
Ordering InformationScreening size kits and components (bulk and custom) available with special pricing. Please call. To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products. Choose "Order Online" in table to see pricing and order online (U.S. only). | Buy | Product # | Description | Pkg. Size | | Order Online | 8402901 | Phospho-H2AX Activation Kit | 1 x 96 | | Order Online | 8402902 | Phospho-H2AX Activation Kit | 5 x 96 |
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