Informações para Pedidos
H2A.X Phosphorylation & p53 DNA Damage Assay
Description:
H2A.X Phosphorylation & p53 DNA Damage Assay
Product Overview:
Millipore’s HCS225 assay provides a complete solution for identifying and quantifying phospho-histone H2A.X(Ser139) and p53 in cellular imaging studies. The reagents in the kit have been specifically optimized for HCS applications. The assay is designed to enable visualization and quantitative detection of phosphorylated histone H2A.X and p53, allowing the characterization of compounds that may induce or inhibit cellular injury such as DNA damage or oxidative stress. The nuclear dye (Hoechst 33342) may be used for measurements of cell number, DNA content and nuclear size. Additionally, the assay can be multiplexed with other probes, e.g., for apoptotic pathway studies, oncology drug efficacy trials or in vitro toxicology applications.
Background Information:
H2A.X
One of the principal responses to DNA damage resulting in double-stranded DNA breaks (DSBs) is the activation of the ATM-initiated signaling cascade to arrest cell division until repairs can be made. Agents that cause DSBs include oxidative stress, ionizing radiation, topoisomerase inhibitors, and DNA binding drugs. A major substrate of the kinases in this cascade is H2A.X. H2A.X is a variant isoform of the histone H2A protein, containing several serine residues (including serine 139) in its unique carboxy-terminal amino acid sequence that are rapidly phosphorylated in response to DSBs. H2A.X is rapidly phosphorylated at serine 139 by the ATM kinase in response to even a few DSBs, and accumulates at the sites of DNA damage forming distinct nuclear foci. These events are important for recruitment and maintenance of the DNA repair machinery to the site of the break. Thus the presence of phospho-histone H2A.X (Ser139) is a reliable marker of DSBs and DNA damage. As DNA repair progresses, the number of foci declines.
p53
Since its discovery in 1979, the tumor suppressor protein p53 has maintained a prominent role in cancer research and drug discovery. p53, often termed ‘guardian of the genome’, integrates diverse physiological signals in mammalian cells and is central to many anti-cancer mechanisms, regulating expression of genes involved in growth arrest, apoptosis, cellular senescence and repair of DNA damage. p53 lies at the hub of complex signaling networks that can integrate a multitude of stress signals. In normal cells p53 is present at low levels, inactive and bound to the protein MDM2 which prevents its action and promotes its degradation. When the cell is confronted with stressors, including, but not limited to, DNA damaging agents (e.g. UV radiation, certain drugs), oxidative stress, mitogenic signals, metabolic alteration and activated oncogenes, p53 accumulates in the nucleus, where it becomes activated. Upon activation, p53 can act as a potent transcriptional activator or repressor, translocate to the mitochondria to induce apoptosis, or localize directly to sites of DNA damage and promote repair.
One of the principal responses to DNA damage resulting in double-stranded DNA breaks (DSBs) is the activation of the ATM-initiated signaling cascade to arrest cell division until repairs can be made. Agents that cause DSBs include oxidative stress, ionizing radiation, topoisomerase inhibitors, and DNA binding drugs. A major substrate of the kinases in this cascade is H2A.X. H2A.X is a variant isoform of the histone H2A protein, containing several serine residues (including serine 139) in its unique carboxy-terminal amino acid sequence that are rapidly phosphorylated in response to DSBs. H2A.X is rapidly phosphorylated at serine 139 by the ATM kinase in response to even a few DSBs, and accumulates at the sites of DNA damage forming distinct nuclear foci. These events are important for recruitment and maintenance of the DNA repair machinery to the site of the break. Thus the presence of phospho-histone H2A.X (Ser139) is a reliable marker of DSBs and DNA damage. As DNA repair progresses, the number of foci declines.
p53
Since its discovery in 1979, the tumor suppressor protein p53 has maintained a prominent role in cancer research and drug discovery. p53, often termed ‘guardian of the genome’, integrates diverse physiological signals in mammalian cells and is central to many anti-cancer mechanisms, regulating expression of genes involved in growth arrest, apoptosis, cellular senescence and repair of DNA damage. p53 lies at the hub of complex signaling networks that can integrate a multitude of stress signals. In normal cells p53 is present at low levels, inactive and bound to the protein MDM2 which prevents its action and promotes its degradation. When the cell is confronted with stressors, including, but not limited to, DNA damaging agents (e.g. UV radiation, certain drugs), oxidative stress, mitogenic signals, metabolic alteration and activated oncogenes, p53 accumulates in the nucleus, where it becomes activated. Upon activation, p53 can act as a potent transcriptional activator or repressor, translocate to the mitochondria to induce apoptosis, or localize directly to sites of DNA damage and promote repair.
Key Applications:
- Immunofluorescence
- in vitro Toxicology Assays
- High Content Analysis
- Immunocytochemistry
Application Notes:
DNA Damage and Repair
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Kit or Assay Type:
- High-Content Screening
- High-Content Analysis
Configuration:
Sufficient reagents for 5 X 96 well plates
Components:
- 1. Mouse Anti-Phospho-Histone H2A.X (Ser139) HCS Primary Antibody, 100X
- 2. HCS Secondary Antibody (donkey anti-mouse IgG, FITC conjugate), 200X
- 3. Sheep Anti-p53 HCS Primary Antibody, 100X
- 4. HCS Secondary Antibody (donkey anti-sheep IgG, Cy3 conjugate), 200X
- 5. Hoechst HCS Nuclear Stain, 200X
- 6. HCS Fixation Solution with Phenol Red, 2X
- 7. HCS Immunofluorescence Buffer, 1X
- 8. HCS Wash Buffer, 1X
- 9. Etoposide (S/G2 Arrest Control Compound), 250X
- 10. Camptothecin (Control Compound), 250X
- 11. DMSO for Compound Serial Dilution
- 12. Compound Dilution Buffer
- 13. Plate Sealers
Storage Conditions:
Store kit components under the conditions indicated on the labels. Discard any remaining reagents after 6 months.
Detection Methods:
Fluorescent
Materials Required but Not Delivered:
1. Sterile, tissue culture-treated black/clear bottom microplates suitable for High-Content Imaging.
2. Cell-type for assay, e.g., HeLa (human cervical adenocarcinoma, ATCC #CCL-2) or A549 (human lung carcinoma, ATCC #CCL-185) or HepG2 (human hepatocellular carcinoma, ATCC #HB-8065).
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. Imaging system must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.
2. Cell-type for assay, e.g., HeLa (human cervical adenocarcinoma, ATCC #CCL-2) or A549 (human lung carcinoma, ATCC #CCL-185) or HepG2 (human hepatocellular carcinoma, ATCC #HB-8065).
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. Imaging system must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.