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Faster protein analysis with pg/µL sensitivity and expanded linear range

By Martin Greiner
Agilent Product Manager, 2100 bioanalyzer

Protein quality assurance/quality control (QA/QC) requires excellent sensitivity, a wide linear dynamic range, and reproducible quantification. Traditional methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis are very time-consuming and lack either reproducible quantification or throughput. The new Agilent High Sensitivity Protein 250 kit for the Agilent 2100 bioanalyzer enables reliable protein sizing and quantification while eliminating drawbacks of traditional methods. It offers:

• Sensitivity down to low pg range – which is better than SDS-PAGE with silver stain
• Four orders of linear dynamic range for quantification
• A 30-minute runtime for 10 samples
• A pre-validated method that includes chips and reagents
• Sample consumption of only 5 µL
• Minimal waste
• No exposure to hazardous materials

The bioanalyzer approach with electrophoretic separation of proteins on a small glass chip provides automated measurement and digital data. It is faster and easier than the slow, manual SDS-PAGE/silver staining process, and provides an integrated solution that is user-independent and more reproducible.

New approach increases sensitivity and linear dynamic range

Scientists in protein research need a tool that can measure both small amounts of impurities and major protein products in a single quantitative run. To achieve sufficient sensitivity for the impurity, traditional determinations often require you to overload the run with the major compound. This approach risks either poor quantification for the main compound if it is measured close to detector saturation, or less reliable analysis of the minor compound if it is measured close to the noise level. To achieve both a wider linear dynamic range for quantification and greater sensitivity, Agilent scientists recently developed a new approach for the 2100 bioanalyzer.

	Figure 1. Workflow from sample preparation through generation of digital data is faster than traditional methods. (Click here to see this image larger.)

The original protein kits for the 2100 bioanlyzer used an intercalating dye that binds to the SDS coating of the proteins during analysis. This method delivers very homogenous quantification for various types of proteins, but the high background of the dye in the gel matrix limits sensitivity to around 5 ng/µL (which is comparable to typical Coomassie stains in slab-gels).

To circumvent this problem for the High Sensitivity Protein 250 kit, Agilent developers implemented a covalent labeling reaction that is done separately from the on-chip protein separation and detection. The reaction uses N-hydroxy-succinimidyl (NHS) ester chemistry to modify lysine residues of proteins. Because protein staining is done off-chip, no dye is added to the gel matrix. The result is a very sensitive measurement that is nearly background-free. Figure 1 shows the new workflow.

	Figure 2. In this example, the Agilent 2100 bioanalyzer with the High Sensitivity Protein 250 kit delivers better sensitivity than standard silver-stained SDS-PAGE, and provides sufficient linearity to measure both the main peak and an impurity at the 0.05% level. (Click here to see this image larger.)

Figure 2 shows a separation of seven standard proteins in a direct comparison of SDS-PAGE/silver stain versus the new High Sensitivity Protein 250 kit on the 2100 bioanalyzer. With SDS-PAGE, the limit of detection is roughly reached in the lane with a total protein load of 6 ng (0.9 ng/protein). With the new assay for the 2100 bioanalyzer, well-defined bands for all proteins are still visible with a total load of 0.5 ng (0.07 ng/protein). The most exciting result is the linear range of four orders of magnitude, which enables reliable measurement of both the main peak and an impurity down to a level of 0.05%.

Many advantages over traditional methods

The new Agilent High Sensitivity Protein 250 kit for the 2100 bioanalyzer offers a fast and flexible way to achieve silver-stain sensitivity or better. By avoiding the poor reproducibility and comparability of SDS-PAGE, the Agilent 2100 bioanalyzer provides a valuable tool for both method development and QA/QC labs. Unlike slab gel techniques, the 2100 bioanalyzer delivers reliable quantification in a digital format that can easily fulfill Good Laboratory Practice (GLP) and 21 CFR Part 11 compliance. And compared with standard capillary electrophoresis methods, this Agilent solution provides the advantages of a pre-validated method and a throughput of 10 samples in 30 minutes. For more information about the Agilent High Sensitivity Protein 250 kit, visit our product page.