Bio-Rad Experion Protein Analysis Kits - Manual (Page 36)

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5.4 Performing Absolute Quantitation
Experion software also allows quantitation using a calibration curve generated by a range of known
concentrations of that protein (absolute quantitation). In this method:
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Different concentrations of the protein are separated in different sample wells
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The peak areas of the different concentrations are compared to the peak area of an internal
standard. As with relative quantitation, either the upper marker or a user-defined protein
can be used as the internal standard
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The protein concentrations in the samples are then determined from this calibration curve
Experion software displays the results for both relative and absolute quantitation, enabling easy
comparison of the accuracy achieved by these different approaches.
5.4.1 Absolute Quantitation Using the Upper Marker as Standard
1. Prepare a dilution series of 3–6 different concentrations of the protein used for calibration. The total
protein concentration in each sample must be within the linear dynamic range of the assay. Prepare
these samples with the other samples for Pro260 analysis as described in Section 3.5.
2. In the Run Info tab under Sample Information, designate the wells that contain the proteins for
the calibration curve by selecting them in the Use as Standard column (Figure 5.4). Enter their
concentrations in the Standard Concentration column and click Apply.
3. Run the Pro260 analysis.
Fig . 5 .3 . Results from
relative quantitation.
Fig . 5 .4 . Run Info tab illustrating the creation of a 5-point calibration curve with wells 1–5 .
Experion Automated Electrophoresis System