Bio-Rad Experion Protein Analysis Kits - Manual (Page 55)

Data Analysis: Normalization, Sizing, and Quantitation
Following separation, Experion software analyses proteins using one or more of the following:
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Internal markers for normalizing the migration times of samples in the different wells
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Pro260 ladder for determining fragment size (sizing)
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Internal markers or calibrants for determining concentration (quantitation)
Normalization: Aligning the Protein Peak Migration Times
To compensate for small variations in each separation, Experion Pro260 analysis uses internal markers
to normalize the migration times among samples. The two internal markers, an upper marker (260 kD)
and lower marker (1.2 kD), are included in the Experion Pro260 sample buffer. Therefore, both of these
markers are added to each sample and the Pro260 ladder (Figure A.1). Inclusion of these markers and
the normalization process ensure that the software accurately identifies and sizes peaks.
Protein Sizing: Determining Molecular Mass
The first sample analyzed is the Pro260 ladder, a modified version of the Precision Plus Protein ™
standards that has been optimized for automated electrophoresis on the Experion system. The Pro260
ladder contains nine purified recombinant proteins of 10–260 kD. Experion software constructs a
standard curve of migration time as a function of size from the ladder separation. It then calculates the
size of the proteins from the sample wells by comparing their migration times to the curve.
Protein Quantitation: Determining Protein Amount
Experion software offers two different types of protein quantitation methods: percentage determination
and concentration determination. All measurements are based on the time-corrected peak area
(corrected area) of each peak identified in an electropherogram. The corrected area of a peak is
proportional to the amount of the protein it represents in a mixture. For more details of the protein
quantitation methods used by Experion software, refer to bulletin 5784.
Percentage determination — measures the percentage of each protein in a mixture (% total). This
method is commonly used for determining protein content, protein purity, and protein stability. The
accuracy of this method depends on the dye binding efficiency of each component in the protein
mixture. As with other dye-based assays or other methods of quantitation using SDS-PAGE, differences
in the amino acid sequences or structures of proteins result in their unique interaction with the Pro260
stain-LDS micelle complexes, which in turn affects peak intensity. As a result, the % total value may not
always reflect the actual percentage in mass for each protein in a mixture. If the component is well-
characterized, this method of quantitation can be an efficient method for processes requiring routine
monitoring of protein samples, such as protein manufacturing and purification
Concentration determination — provides the amount of the protein(s) in a mixture rather than just
a percentage of the total. There are several different ways that concentration determination can be
performed, and these methods provide more or less accuracy depending on the type of internal
standard used and the extent to which a calibration curve is used:
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Relative quantitation — Experion software uses an internal standard to estimate the
concentrations of sample proteins. Estimates are calculated by comparing the corrected
peak area of the sample peak to that of the internal standard in each sample well. The
internal standard can be either the upper marker (260 kD) or a user-defined internal
standard (between 10 and 260 kD) added to each sample at a known concentration
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Absolute quantitation — Experion software can also create a calibration curve to use for
quantitation. To create the calibration curve, a protein calibrant at different concentrations is
separated in different sample wells. The corrected areas of the different sample peaks are
compared to that of an internal standard (as with relative quantitation, the upper marker or a
user-defined protein can be used). Experion software plots the resulting ratios as a function
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Experion Pro260 Analysis Kit