I am doing it with the sigma GST assay kit. My blank buffer consisting of PBS, GSH and cDNB is also giving an absorbance. So can you just give me a detailed description in case am making a mistake.
Hi Swati Gupta,
This is perfectly normal. GSH and CDNB do react spontaneously (i.e. in the absence of transferases). As you can see, the enzyme solution increased the rate in comparison to the assay without enzyme. Then, you just have to subtract it. To reduce the rate of the spontaneous reaction it is a common practice to user mildly acidic pH (6.5).
Take a look at the classical reference for the method:
Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139.
Other good reference:
Rice-Evans, Ziplock and Symons (1991) Techniques in free radical research. Elsevier.
An example:
Pannunzio TM, Storey KB (1998) Antioxidant defenses and lipid peroxidation during anoxia stress and aerobic recovery in the marine gastropod Littorina littorea. J Exp Mar Biol Ecol 221:277–292. doi: 10.1016/S0022-0981(97)00132-9
Hi
I think following paper may be helpful;
Effects of phenethylisothiocyanate on the expression of glutathione S-transferases and hepatotoxicity induced by acetaminophen
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