I'm trying to detect the GST activity in liver of goldfish. I chose CDNB as my substrate. But when I measured OD of the blank solution which including 2.8ml of 0.1M PB (pH 6.5), 0.1ml of 30mM GSH and 0.1ml of 30mM CDNB at 340nm, I found the absorbance was beyond the highest detected limitation of the spectrophotometer. That means the substrate has intensive background absorption. Normally the absorption of the blank solution is nearly to zero. When I decreased the concentration of the substrate, the OD decreaed too. However I diluted the CDNB solution 100 dilutions, the OD was still higher than 1.0.
I bought CDNB and GSH from Sigma, and dissolved CDNB in ethanol while GSH in 0.1M PB (pH 6.5). I measured OD of the blank solution on two different spectrophotometers, so it was not the problem of the instruments. I don't know why, how to deal with the problem. Anyone can help me?
Looking forward to the replies. Thank you!