I am working on pectinase enzyme assay. I incubated 900 ul of substrate for 10 minutes in the water bath, followed by adding 2ml of DNSA reagent, then 100ul of enzyme extract added finally i read the absorbance @ 540 OD. However the values are high. How can I troubleshoot high enzyme blank values in Pectinase assay ?
Now I understand what your problem is. The dinitrosalicylic acid reagent reacts with glucose to give a colored reagent. It is best to read the absorbance quickly since the reagent becomes darker with time. So it is best to cool the tubes in a water bath.
I believe that your substrate contains free glucose which is why you get a high absorbance for your blank. You can either use the blank to zero the spectrophotometer (since you want the difference between the blank and the reacted solution), or you can find a different pectin which has less glucose in it.
Since there is some variability in the reaction, it is best to do everything in duplicate or triplicate (including the blanks).
This web page uses the wavelength of 575 nm. This might help with the intensity.