Step 1 is to construct a standard curve that defines the relationship between the known concentrations of a
substance and a measured value such as optical density, fluorescent intensity, radioactivity, etc. The results are
graphed with the known concentrations on the X axis and the measured variable on the Y axis.
Step 2 is to fit a curve or line to the data and determine the concentrations of the unknown samples.
Steps 3-5 are to format the graph to make it more informative.
Step 6 is to insert the results of the interpolation in a table on the chart.
Prism can fit standard curves using nonlinear regression (curve fitting), linear regression, or a cubic spline (or
LOWESS) curve. This example uses linear regression and will take approximately 30 minutes to complete,
resulting in this graph:
Protein standard curve
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Optical Density
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micrograms
If you didnt pick the sample data set, enter data for the standard curve (shown below). In the sample data,
rows 1-7 are for the standard curve, where you know both X and Y. The Y column of rows 8-10 has the optical
density of the unknown samples. The corresponding X values are intentionally left blank. Those are the values
we need to figure out.
1
Click twice on the default sheet name in the Prism Navigator tree to rename the sheet.
View the graph that is produced from the data by clicking Protein Standard Curve under Graphs in the
navigator bar. In the Change Graph Type dialog box that appears, select Points only (highlighted by the red
arrow):
2
The graph of this data should now look like this:
Protein standard curve
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Optical Density
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micrograms
Note: This Interpolate analysis is new to Prism 6. You could also do the interpolation using the
linear or nonlinear regression analyses, but this new Interpolate analysis is more focused and
thus easier to use.
In the Parameters: Interpolate a Standard Curve dialog box, select Line. Uncheck the box next to Plot
curve with 95% confidence band. These selections are highlighted below with red arrows.
3
The graph of this data should now look like this:
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Optical Density
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micrograms
Prism performs the fit and displays the outcome in sheets under the Results heading. The Interpolated X
values sheet listed under this analysis of the Results to view the unknown values computed based on the
linear regression. Prism reports the corresponding X value for each unpaired Y value on the data sheet.
4
Switch to the Table of results sheet (shown below). This sheet provides the best-fit values from the analysis:
5
After you have made these changes, the graph includes the unknown values:
Protein standard curve
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Optical Density
(Entered)
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Optical Density
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micrograms
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6
Step 5: Replace the unknown data points with spikes
Instead of representing data using points, data can also be represented as spikes or bars projecting from the X
axis. To do this, double click on one of the symbols on the graph. In the Format Graph dialog box, select
Linear reg. of Protein standard curve: Interpolated X values (red box) and check the box next to
Show bars/spikes/droplines. Set the Width to 1 and select the Color and Border color and Border
thickness. These settings are highlighted by the red arrows below:
The final graph should now look like shown below. Note that the table is live. If you change the data, and so
change the interpolated results, the table will update automatically.
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Optical Density
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micrograms