Absolute quantitation with the Agilent 2100 Bioanalyzer and the Protein 200 LabChip® kit. The Protein 200 LabChip® kit allows sizing and quantitation of proteins ranging in size from 14 to 200 kD. Relative quantitation is performed in comparison to the upper marker, used as internal standard in each sample. However, the accuracy of the relative quantitation is affected by the staining efficiency of the protein dye. Absolute quantitation is enabled with the Agilent 2100 bioanalyzer software (revision A.02.01) using the Protein 200 assay in combination with protein standards of known concentrations. Absolute quantitation improves quantitation accuracy by eliminating the differences in staining efficiency among proteins. This Application Note describes how to perform absolute quantitation with the Protein 200 assay and demonstrates the obtained improvement in accuracy.
The Protein 200 LabChip kit enables sizing and quantitation of proteins ranging from 14 to 200 kD. While relative quantitation uses the upper marker (myosin) as an internal standard, its accuracy is affected by varying staining efficiencies among different proteins. Absolute quantitation addresses this by using a calibration curve generated from standards of known concentration for the specific protein of interest.
| Protein Size Range | 14 to 200 kD |
|---|---|
| Software Revision | A.02.01 |
| Relative Quantitation Reproducibility | approximately ±30 % |
It eliminates differences in staining efficiency among proteins by using a calibration curve generated with identical proteins of known concentrations.
The Protein 200 upper marker, which is myosin, is used as the internal standard in each sample.
Yes, BSA or another protein can be substituted to generate a calibration curve, though this results in relative quantitation rather than true absolute quantitation.