Description
Product Usage Information
Boil for 3 min prior to use. Load 10 µL of phosphorylated and nonphosphorylated DNA Damage Control Cell Extracts per lane.
Storage
Supplied in SDS Sample Buffer: 62.5 mM Tris- HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red. Store at -20°C, or at -80°C for long-term storage.
Background
Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases are PI3K-related kinase (PIKK) family members that phosphorylate multiple substrates on serine or threonine residues that are followed by a glutamine in response to DNA damage or replication blocks (1-3). p53 is phosphorylated by ATM, ATR, and DNA-PK at Ser15. This phosphorylation impairs the ability of MDM2 to bind p53, thereby promoting accumulation and activation of p53 in response to DNA damage (4,5). Chk1 and Chk2, downstream protein kinases of ATM/ATR, play an important role in DNA damage checkpoint control, embryonic development, and tumor suppression (6). Chk1 is phosphorylated at Ser280 and Ser296 following DNA damage. The amino-terminal domain of Chk2 contains a series of seven serine or threonine residues, including Thr68, each followed by glutamine (SQ or TQ motif). After DNA damage by ionizing radiation (IR), UV irradiation, or hydroxyurea treatment, Thr68 and other sites in this region become phosphorylated by ATM/ATR (7-9). The breast cancer susceptibility proteins BRCA1 and BRCA2 are frequently mutated in cases of hereditary breast and ovarian cancers and have roles in multiple processes related to DNA damage, repair, cell cycle progression, transcription, ubiquitination, and apoptosis. Numerous DNA-damage-induced phosphorylation sites on BRCA1 have been identified, including serine 1524, and kinases activated in a cell cycle-dependent manner, including Aurora A and CDK2, can also phosphorylate BRCA1. IR, DNA, and radiometric-induced DNA damage also result in rapid phosphorylation of the histone H2A family member H2A.X at Ser139 by ATM (10,11). Within minutes following DNA damage, Ser139-phosphorylated H2A.X localizes to sites of DNA damage at subnuclear foci (12).
Alternate Names
DNA damage; extract; lysate
