Techniques include: 
  • measurement of thiols and antioxidant enzyme activity using activity assays, activity gels, spectrophotometric assays and HPLC assays; 
  • measurement of immunoreactive protein for antioxidant enzymes using western blotting; 
  • measurement of steady-state mRNA levels for antioxidant enzymes using PCR analysis; 
  • determination of antioxidant enzyme gene copy number and gross chromosomal changes; 
  • transfection and characterization of cell lines expressing antioxidant enzyme sense and antisense cDNAs; maintain, distribute, and provide a repository for reagents used in the study of antioxidant proteins: i.e., antibodies, cDNAs, expression  vectors, siRNA reagents and cell lines; 
  • provide expertise and equipment for studying a range of O2 tensions from radiobiological hypoxia (<0.1% O2) to physiologically relevant tissue O2 tensions (4-6% O2) in tissue culture experiments measuring clonogenic cell survival. 
The following enzyme assays are routinely available: 
  • copper-and zinc-containing superoxide dismutase (CuZnSOD), 
  • manganese-containing superoxide dismutase (MnSOD), 
  • catalase (CAT), glutathione transferases (GSTs), 
  • glutamyl transpeptidase (GGT), 
  • glutamylcysteine synthetase (GCS), 
  • glutathione reductase (GR), 
  • glucose-6-phosphate dehydrogenase (G6PD), 
  • thioredoxin reductase (TRR), and glutathione peroxidases (GPx). 
  • Assays for the detection of prooxidant production in living cells (superoxide, hydrogen peroxide, lipid peroxidation products, etc.) are also be available. 
  • Antibodies, cDNA probes, lentiviral vectors, transplantable human tumor xenograft models, will be provided. 

The AEC will also aid in transfecting conditional over expression cDNAs and lentiviral vectors into cell lines of interest to investigators and confirmation of expression. Services to measure all five-electron transport chain (ETC) complex activities, TCA cycle enzymes including total aconitase and citrate synthase, glutathione/glutathione disulfide, thioredoxin/thioredoxin disulfide, glucose, ATP, lactate, NADP+/NADPH and NAD+/NADH are also available for monitoring oxidative metabolism. AES staff will carry out the proposed work or will advise the user and staff on how to perform and analyze the experiments in their own labs.