How do I calculate values from a standard curve?
Introduction
A standard curve is used to accurately determine the concentration of your sample from the signal generated by an assay. The signal is never perfectly proportional to the sample concentration. A standard curve is designed to correct for these effects, so you know which concentration a given signal value corresponds to.
Standard curves are generally used when you need quantitatively accurate results. The general principles described below apply to our cell-based, biochemical and protein activity assays, as well as our ELISA kits.
Specific instructions for your kit can be found in the protocol booklet's 'Data Analysis' section.
Performing a standard curve
Specific instructions for standard curves will be indicated in the relevant protocol booklet under the ‘data analysis’ section
1. Check the protocol booklet to determine whether a standard curve is required for your kit
- Standard curves are usually required if you're looking to use the kit for quantitative measurement of concentration (rather than semi-quantitative or qualitative analysis).
2. Measure the signal on a range of known concentrations of a standard
- Concentrations to use will be indicated in the protocol booklet
- Note that it's often advised to perform this step in duplicate or triplicate for greater reliability
3. Plot concentration used on the x-axis vs signal on the y-axis (examples below)
4. Analyze the plot to determine the trend
- Specific instructions for analysis will be found in the protocol booklet under ‘data analysis’ section.
- ELISA data (which is often sigmoidal) usually uses a four parameter curve fit (4PL), although other models can be used if they give a better fit.
- Many other kits show approximately linear behaviour, so a linear trendline is suitable.
5. Run the assay on your target sample and measure the signal
- To be valid, the signal you get should be within the range of values obtained from the standard curve. If not, dilute or concentrate your samples as needed.
6. Use the trend from the standard curve to calculate the concentration from each signal!
- Follow specific instructions in the protocol booklet for your kit.
- Make sure all samples are within the range of the standard curve. If signals are outside this range, the sample will need diluting or concentrating as appropriate.