To allow for the differences in yellow color in each plant extract primarily due to varying amounts of contaminating photosynthetic pigments, it was necessary to blank each extract separately with diluted extract in buffer lacking the GUS substrate, PNPG. Once you have obtained corrected absorbance values by subtracting the absorbance due to yellow color that is not related to PNPG breakdown, you also must normalize the activities further so that you can compare plants. Typically activities are expressed on a per mg fresh weight or protein basis. Because the method used to cut the tissue disks produce tissue pieces of a uniform size we will express our enzyme activities on a per disk basis. Calculate the GUS activity found in each of your extracts as: Activity = nmoles product formed/minute-leaf disk. You may then compare your data with those of your classmates.
Structural Evidence for Transgenic Plants
First calculate the nmoles product formed/ reaction. The molar extinction coefficient of p-nitrophenol is 14,000. Therefore an absorbance of 0.01 represents 1 nmole of product formed in your 1.4 ml reaction volume (assay buffer + extract + stop buffer).
It follows that if the corrected A415 is the absorbance after subtracting the color blank absorbance from the activity absorbance then:
A415 of assay – A415 extract blank = corrected A415
corrected A415/0.01 = nmoles product (PNP) formed/reaction
To calculate the nmoles PNP formed/ min leaf disk:
(corrected A415/0.01)×(1/0.1 ml extract)×(1/? min)×(1.5 ml leaf extract/5 leaf discs) = nmol PNP/min-leaf disc
Assaying the Transgenic Plants (HOME)
Leaf Extract Preparation
Spectrophotometric Assay for GUS activity
GUS Activity Assay by Histochemistry