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 ERDC TN-DOER-C30
February 2003
14) Add 50 L of ONPG to each well and place plate in the temperature-controlled
plate reader.
15) Monitor the reaction in the 405- to 420-nm range, taking readings every minute
for at least 15 min.
Check the kinetics of each well to be sure that the reaction remained linear over
the time course; if the reaction proceeds at too fast a rate, the rate will plateau
and the overall average mOD/min will be lower than the maximal rate during
the linear phase.
16) Print out the data report (should give a rate of reaction for each well, in mOD per
minute) and save the data files.
17) Remove the plate, exit the program, then shut down the computer and plate reader.
V.
DATA PROCESSING
The data generated from the plate reader (mOD/min) is used to generate a log-dose response
curve for both the Ponasterone A alone and the Ponasterone A with the sample. Using a graphics
package such as SigmaPlot, a sigmoidal regression can be generated and an EC50 and maximal rate
determined for each curve. Significant shifts in either parameter indicate that the environmental
sample is interacting with the receptor-ligand complex (EcR receptor-Ponasterone A complex).
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