Bio-Rad Experion Protein Analysis Kits - Manual (Page 38)

34 Technical Support: 1-800-4BIORAD • 1-800-424-6723 • www.bio-rad.com
5.4.2 Absolute Quantitation Using a User-Defined Internal Standard
This is the most stringent method of quantitation. It combines two of the approaches described above:
n
A calibration curve is generated using known concentrations of a protein, and
n
A user-defined protein is used as the internal standard
1. Prepare a dilution series of 3–6 concentrations of the protein that you will use for calibration.
2. Add a known concentration of the internal standard to each sample (except the Pro260 ladder),
including those that will be used for calibration. Prepare these samples and other samples as
described in Section 3.5.
3. Under the Run Info tab, designate the wells that contain the proteins for the calibration curve and
enter their concentrations (Figure 5.5). Click Apply and run the Pro260 analysis.
4. After evaluating the run as described in Section 3.10, select Analysis > Internal Std . and Std
Protein Calibration Curve .
5. In the Internal Std . and Std . Protein Calibration Curve window (Figure 5.5), do the following:
a. Under Standard Information, next to Standard Protein Molecular Weight, enter the MW
corresponding to the region where the software searches for a peak, for both the calibrants
(standards) and the samples. The software checks for a peak in the sample wells that has the
entered value (± 5%). If necessary, modify the standard concentrations by double-clicking on the
concentration and entering a new value.
b. Select Use User Internal Standard and enter the name, concentration, and size of the internal
standard. Click Apply.
c. Under Calibration Curve Settings, if the protein used for calibration (calibrant) is different or
has a different mass than the protein being quantitated, select Quantitate All Proteins and click
Apply. If you do not do this, the software only reports absolute concentrations for peaks that are
the same size as the calibrant.
d. Under Calibration Curve Settings, select the Linear Regression to be used. Click Apply to
update the calculation.
6. The calibration curve appears, displaying the equation for the linear regression. The table below the
graphic displays the data for the protein standard samples. Click Close.
7. In the Results table, the concentration for each protein derived by relative quantitation against
the user-defined internal standard appears in the Concentration column, and the calibrated
concentration (using the calibration curve in conjunction with the user-defined standard) appears in
the Calib . Conc . column.
Experion Automated Electrophoresis System