DNA Damage Assay Human bronchial epithelial cells (hBE)

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

Get tips on using OxiSelect™ Intracellular ROS Assay Kit (Green Fluorescence) to perform ROS assay cell type - PC12

Get tips on using Amplex™ Red Hydrogen Peroxide/Peroxidase Assay Kit to perform ROS assay cell type - C2C12

Get tips on using AllPrep DNA/RNA/miRNA Universal Kit to perform RNA isolation / purification Tissue - mouse adipose tissue

Get tips on using Dual-Light™ Luciferase & β-Galactosidase Reporter Gene Assay System to perform Reporter gene assay β-galactosidase substrates - Hep3B

Get tips on using Senescence Cells Histochemical Staining Kit to perform Cell cycle assay mouse - L929

Get tips on using Mitochondrial ROS Activity Assay Kit (Deep Red Fluorescence) to perform ROS assay cell type - mouse cardiomyocytes

Get tips on using Apo-ONE® Homogeneous Caspase-3/7 Assay to perform Apoptosis assay cell type - SH-SY5Y

ROS has a very short half-lives in biological environment as they are influenced by exposure to ambient oxygen. As it is highly reactive and hard to measure care should be taken to ensure the stability of the sample during isolation, preparation, storage, and analysis.