In-Vitro Histone H2AX Phosphorylation Assay

Theoretical Background / Context

• In genetics, genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. 
• While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic. 

The alteration can have direct or indirect effects on the DNA:

• The induction of mutations mistimed event activation, and direct DNA damage leading to mutations. 
• The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. 
• Cells prevent expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.

Principle of Work

• DNA damage response is crucial to maintain the homeostasis of cells. 
• Damage that remains unrepaired or incorrectly repaired may lead to genetic mutations, instability, and increased risk of carcinogenesis. 
• One of the most serious sources of damage in cells, DNA double-strand breaks (DSBs) are often induced by various sources, including ionizing radiation and exposure to DNA- damaging chemical or environmental stress. 

• On the occurrence of DSB, cells initiate DNA response signalling and recruit DNA damage repair proteins to affected DNA sites to repair the altered DNA. 
• After the formation of DSBs, H2AX, a variant form of histone H2A and is ubiquitously distributed throughout the genome, is rapidly phosphorylated on a serine 139 residue to create γH2AX through h2ax phosphorylation dna damage mechanism demonstrating how to phosphorylate a protein in vitro.
• The γH2AX is phosphorylated in megabase regions surrounding the DNA break site. 
• Large numbers of γH2AX molecules can be visualized as foci in the nuclear region by immunostaining with antibodies that recognize γH2AX. 
• Monitoring of γH2AX foci formation by a fluorescent microscope is useful for detecting the incidence of DSBs.