Competition:Analysis of Data:Sample Experiment
Analysis of Data and Interpretation of Results from Sample Experiment
In this section the data generated from the sample competition experiment will be
analyzed. This section assumes that you know how to plot these graphs
and
determine IC50 valuesas described on the previous two pages. You
will be lead through the following steps:
- In the first step you will be asked to convert the concentration of prazosin added into log units.
- The data will be plotted three ways: Bound (cpm) versus - Log of Prazosin, % of
Control versus the - Log of Prazosin, and % of Specific Bound versus the - Log of
Prazosin. These plots were explained in the previous section.You will be
asked to calculate the values needed to plot the data.
- You will be asked to estimate the IC50 values from each plot. You will be given the results of the nonlinear regression analysis of the data.
You will be asked to calculate the Ki value from the IC50 value. To do this you will be asked to calculate the concentration of [3H]RX821002 actually present in the assay tubes.
Click here to review the purpose of sample experiment.
Analysis of Data as Average Bound (cpm) versus the - Log of Prazosin
- The following table lists the concentrations of prazosin that were used in the sample experiment. Please convert the prazosin concentration Molar units (Use E to represent the exponential value of 10. For example 10-9 = E-9). Next covert the molar units to log (M) units and place the answers in the following table.
The Bound (cpm) values obtained from the 12 sets of duplicate tubes are given in the table below. Matthew has entered the results of your calculations of Log of Prazosin in the first column. Note the 0 concentration of prazosin has been converted to Log -12. This value is > 10 fold lower than than the lowest concentration of prazosin used. It simplifies plotting the data on the graph. Please calculate the Average Bound (cpm) to the nearest whole number and enter the values in the table.
The plot of Bound (cpm) versus - Log of Prazosin looks like this:
Note the following factors on this graph
- The curve shown on the graph was generated using a nonlinear regression analysis for one-site competition using the Prism computer program.
- The first point on the graph is the control value that was obtained by adding the diluent for prazosin. It is difficult to plot the results as 0 concentration as this requires a break in X-axis. One way around this is to express the control value as a concentration of the unlabeled drug that is 10 to 100 fold lower than the lowest concentration of unlabeled ligand. In this instance a prazosin concentration of 10-11 M was chosen to represent the control value.
- The amount of radioligand bound in the presence of 30 然 norepinephrine (NE) is shown. Remember that norepinephrine was used as a marker to tell us where the bottom of the curve should be. The since amount of [3H]RX821002 bound at the lowest concentration of prazosin is similar to that obtained with norepinephrine, it indicates that prazosin is blocking all of the sites that norepinephrine is blocking.
- The top of the curve does not go through the zero control value and is not well defined. The lower end of the curve is well defined. A better designed experiment would be to use prazosin concentrations between 0.1 nM and 3 然.
