#IMAGEJ QUANTIFICATION WESTERN BLOT MANUAL#

H2AX is one of the most conserved variants of the histone H2A and accounts for 2–25% of the H2A protein 21.

#IMAGEJ QUANTIFICATION WESTERN BLOT MANUAL#

In addition, manual or semi-automated assessment of DSBs can be used to elucidate the protective effect of compounds against genomic damage 15, assess the DNA repair process of cells 1, or even establish the endogenous load of DNA DSBs within the cells 16.ĭNA DSBs trigger a DNA damage response that is characterized by activation of DNA repair mechanisms and phosphorylation of the histone protein component H2AX to form phosphorylated H2AX (γ-H2AX) 17, 18, 19, 20. Effective and efficient quantification of DSBs is critical for managing the effective dose for radiation therapy, identifying exposure to cytotoxic agents or environmental factors, and assessing the pharmacodynamics of chemotherapeutics. However, given the detrimental effects of DNA DSBs on cell viability and the induction of mutated cells, it is critical to quantify them effectively to better inform treatment decisions. The ability of different forms of irradiation to induce DNA DSBs has been harnessed in radiotherapy treatments to kill cancer cells 13, 14. Recent studies using a very fast CRISPR Cas 9 system to induce DNA DSBs have tracked their formation over time and length scales 12. Exogenous factors inducing DNA DSBs include cytotoxic chemical agents 2, 3, 4 and environmental 5 and physical factors such as radiation 6, 7, 8, 9 and heat 10, 11. They are difficult to repair and can lead to genotoxicity, cell death, and misrepaired DNA DSBs with potential for neoplastic progression. DNA DSBs present the most serious threat to the cell. Similar content being viewed by othersĭNA damage in cells can be triggered by endogenous mechanisms, exogenous factors, or a combination of both, resulting in a multitude of alterations including DNA base modifications, single-strand breaks, and double-strand breaks (DSBs) 1. These approaches have the potential to improve screening of compounds that either enhance DNA damage or protect against its deleterious effects. Lanthanide based immunodetection of γ-H2AX offers superior detection and a user-friendly workflow. Comparative bulk fluorescent signals achieved only micromolar sensitivity. Time resolved fluorescence detection of europium as a chelated complex enabled quantitative measurement of γ-H2AX foci with nanomolar resolution. Etoposide induced DNA damage in A549 carcinoma cells was compared across all test platforms. For comparison, standard fluorescence detection was employed and analyzed either by bulk fluorescent measurements or by direct foci counting using BioTek Spot Count algorithm and Gen 5 software. The novelty of this work is the application of a time-resolved fluorescence assay using dissociation-enhanced lanthanide fluorescence immunoassay for quantitative measurements of γ-H2AX. Traditional γ-H2AX detection involves counting individual foci within individual nuclei. Phosphorylation of the histone protein H2AX to form γ-H2AX foci directly represents DNA double-strand break formation.

Imagej quantification western blot