Abstract
The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.
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Abbreviations
- 2D:
-
Two-dimensional
- CBB:
-
Coomassie brilliant blue
- BN:
-
Blue native
- B:
-
Buffer (pool)
- BW:
-
Buffer waste (pool)
- S:
-
Sample (pool)
- SW:
-
Sample waste (pool)
- P:
-
Total peak capacity
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Acknowledgments
This project was supported by the National Nature Science Foundation of China (No. 20705005), the Special-Funded Program on National Key Scientific Instruments and Equipment Development (2012YQ04014006), the National Natural Science Foundation of China (21174017), the Cultivation Project for Technology Innovation Program of BIT (2011CX01032), and the Fundamental Research Foundation of BIT (No. 3160012211309).
Conflict of interest
We declare that we have no financial and individual relationships with other people or organizations that can inappropriately influence our work; there is no professional or other personal interest of any nature in any product, service, and/or company, which could be construed as influencing the position presented in, or the review of, this manuscript.
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N. Hou and Y. Chen are co-first authors.
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Hou, N., Chen, Y., Yu, S. et al. Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis. Chromatographia 77, 1339–1346 (2014). https://doi.org/10.1007/s10337-014-2728-3
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DOI: https://doi.org/10.1007/s10337-014-2728-3