The OPA assay Sample Clauses

The OPA assay. An alternative method to measuring thiol content is by reaction with o- phthalaldehyde (OPA). OPA is a dialdehyde which reacts with thiols and primary amines to form a fluorophore. The OPA assay is therefore considered more specific for measuring GSH, which contains both amines and thiols than the DTNB assay151. As OPA reacts with primary amines, protein concentration could affect measurement of thiol concentration. In these experiments, however, cellular thiol content was corrected for protein to avoid this. OPA was purchased from Sigma-Xxxxxxx, UK. OPA is light sensitive; therefore precautions were taken to limit exposure to light throughout protocols. OPA was dissolved in methanol (10 mg/mL) in a foil covered tube. 150 µL phosphate/ETDA buffer (pH 8.0) was added to samples in a black opaque plate before adding 15 µL OPA solution to each well. Plates were then covered in foil and placed in a dark room for 25 mins. A microtitre plate reader was used to measure the fluorescence at 350nm excitation and 420nm emission (Chameleon V, Hidex, Finland).